Regulators of Hedgehog signaling in chondrocytes: Sufu ... · Shu-Hsuan Claire Hsu Doctor of...

Regulators of Hedgehog signaling in chondrocytes: Sufu, Kif7, and primary cilium

By

Shu-Hsuan Claire Hsu

Doctor of Philosophy

Institute of Medical Science

University of Toronto

© Copyright by Shu-Hsuan Claire Hsu 2012

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Regulators of Hedgehog signaling in chondrocytes: Sufu, Kif7, and primary cilium

Shu-Hsuan Claire Hsu

Doctor of Philosophy

Institute of Medical Science

University of Toronto

2012

Abstract

The Hedgehog (Hh) signaling pathway has received attention regarding its important role

in embryonic development, however the mechanism by which pathway regulators, such as

Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh

signaling transduction is not entirely understood. The work presented here examines the roles of

Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they

mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I

show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While

Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively

in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli

complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh

pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive

role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory

function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore,

Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-

independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh

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on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and

primary cilium are dispensable in this process. Although primary cilium is required for Hh

ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional

activity in a cilia-independent manner. The results of this work provide insight into how Hh

signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu

serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte

development.

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Acknowledgments

I sincerely thank my supervisor Dr. Benjamin Alman for his guidance and

encouragement. Enormous thanks to Dr. Chi-Chung Hui for his invaluable suggestions and

guidance. I am also very grateful to my committee members, Dr. David Bazett-Jones and Dr.

Martin Post, for their comments and suggestions. Thanks to Xiaoyun Zhang from the Hui lab for

helping me with the western analysis. Thanks to the entire Alman lab, past and present for their

scientific and moral support. Thanks to my family and friends for their love, for cheering me on,

and their patience with me these past few years. Finally thanks to my boyfriend Robert for his

support and for putting a smile on my face when things did not work at the bench.

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Table of contents

Abstract ..................................................................................................................................... ii

Acknowledgments ................................................................................................................... iv

Table of contents ....................................................................................................................... v

List of abbreviations ................................................................................................................. x

List of figures .......................................................................................................................... xii

Chapter 1 Introduction .............................................................................................................. 1

1.1 Summary ......................................................................................................................... 2

1.2 Overview of growth plate development .......................................................................... 3

1.3 Indian hedgehog (Ihh) and Parathyroid hormone-like hormone (Pthlh) signaling in

growth plate development ........................................................................................................... 4

1.3.1 Ihh/Pthlh feedback loop ............................................................................................ 4

1.3.2 Pthlh-dependent and –independent effects of Ihh .................................................... 4

1.3.3 Actions of Pthlh in the growth plate ......................................................................... 5

1.4 Ihh and Pthlh signaling pathways regulate Gli transcription factors .............................. 7

1.5 Regulators in Hedgehog (Hh) signaling pathway: Sufu and Kif7 .................................. 9

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1.6 The primary cilium in Hh signaling .............................................................................. 11

1.7 Hypothesis & Specific aims .......................................................................................... 14

1.8 Figures ........................................................................................................................... 15

1.9 References ..................................................................................................................... 19

Chapter 2 Kif7 promotes Hh signaling in growth plate chondrocytes by restricting the

inhibitory function of Sufu ........................................................................................................... 25

2.1 Summary ....................................................................................................................... 26

2.2 Introduction ................................................................................................................... 27

2.3 Results ........................................................................................................................... 31

2.3.1 Sufu is differentially expressed in the growth plate and is required for normal

skeletal development ............................................................................................................. 31

2.3.2 Sufu acts as a negative regulator of Hh signaling during chondrocyte

differentiation ........................................................................................................................ 32

2.3.3 Loss of Kif7 in growth plate chondrocytes results in reduced Hh pathway activity

............................................................................................................................................... 33

2.3.4 Sufu-Gli complexes are localized to the ciliary tip in the absence of Kif7 ............ 34

2.3.5 Removal of one copy of Sufu rescues the Kif7 mutant growth plate phenotype .... 37

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2.3.6 Kif7 and Sufu share overlapping functions in Hh signaling during chondrocyte

development........................................................................................................................... 37

2.4 Discussion ..................................................................................................................... 39

2.5 Materials & Methods ..................................................................................................... 44

2.6 Figures ........................................................................................................................... 49

2.7 References ..................................................................................................................... 66

Chapter 3 Sufu mediates the effect of Pthlh on chondrocyte differentiation in the growth plate

...................................................................................................................................................... 70

3.1 Summary ....................................................................................................................... 71

3.2 Introduction ................................................................................................................... 72

3.3 Results ........................................................................................................................... 75

3.3.1 Sufu regulates the effect of Pthlh on chondrocyte differentiation .......................... 75

3.3.2 Kif7 is not required for Pthlh to inhibit chondrocyte differentiation ..................... 76

3.3.3 Pthlh regulates Sufu protein level ........................................................................... 76

3.3.4 Sufu is required for the ability of Pthlh to process Gli transcription factors .......... 77

3.4 Discussion ..................................................................................................................... 79

3.5 Materials & Methods ..................................................................................................... 82

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3.6 Figures ........................................................................................................................... 87

3.7 References ..................................................................................................................... 94

Chapter 4 Conclusions & future directions ............................................................................. 97

4.1 Summary ....................................................................................................................... 98

4.2 Sufu expression is tightly regulated by multiple factors in the growth plate ................ 99

4.2.1 Lessons from genetically modified mice and Pthlh activation ............................... 99

4.2.2 Transcriptional regulation of Sufu ........................................................................ 100

4.2.3 How does Sufu regulate subsequent steps of endochondral ossification? ............ 101

4.2.4 Sufu and osteoarthritis .......................................................................................... 102

4.3 The role of Sufu in Wnt/ß-catenin signaling pathway during chondrocyte development

................................................................................................................................................. 105

4.3.1 Wnt/ß-catenin signaling in skeletal development ................................................. 105

4.3.2 The role of Sufu in mediating Wnt/ ß-catenin signaling ...................................... 106

4.4 Conclusions ................................................................................................................. 109

4.5 Figures ......................................................................................................................... 110

4.6 References ................................................................................................................... 112

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Appendix A: Pthlh negatively regulates Hh signaling activity in chondrocytes through a cilia-

independent mechanism .............................................................................................................. 116

A.1 Summary .................................................................................................................... 117

A.2 Introduction ................................................................................................................ 118

A.3 Results ........................................................................................................................ 121

A.3.1 Ift88ORPK/ ORPK

mice show skeletal defects and dysregulated Hh signaling in

chondrocytes ........................................................................................................................ 121

A.3.2 Pthlh downregulates Col10a1 expression and Hh signaling activity through a cilia-

independent mechanism in chondrocytes ............................................................................ 121

A.3.3 Ciliary tip localization of Sufu is induced by Pthlh ................................................ 122

A.4 Discussion .................................................................................................................. 125

A.5 Materials & Methods .................................................................................................. 128

A.6 Figures ........................................................................................................................ 132

A.7 References .................................................................................................................. 139

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List of abbreviations

APC Adenomatous polyposis coli gene product

ChIP Chromatin immunoprecipitation

Ci Cubitus interruptus

CHX cycloheximide

DAPI 4 ,6-diamidino-2-phenylindole (DAPI)

Gli1 Glioma-associated oncogen family member 1



Gli3FL Full-length of glioma-associated oncogene family member 3

Gli3R C-terminally truncated transcriptional repressor form of glioma-associated oncogene

family member 3

GSK3ß Glycogen synthase kinase 3ß

Hh Hedgehog

H&E Hematoxylin and eosin

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IFT Intraflagellar transport

Ihh Indian hedgehog

IP Immunoprecipitate

Kif7 Kinesin family member 7

Lef Lymphoid enhancer factor

PBS Phosphate-buffered saline

PKA Protein kinase A

Ptch1 Patched 1

Pthlh Parathyroid hormone-like hormone

PTHrP Parathyroid hormone-related protein

PTHR1 Parathyroid hormone 1 receptor

Pur Purmorphamine

Shh Sonic hedgehog

Smo Smoothened

Sufu Supppressor of fused

TCF Transcription factors T-cell factor

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List of figures

FIGURE 1-1 SCHEMATIC REPRESENTATION OF ENDOCHONDRAL OSSIFICATION. ............................. 15

FIGURE 1-2 IHH/PTHLH SIGNALING REGULATION IN THE GROWTH PLATE. ..................................... 16

FIGURE 1-3 HH SIGNALING IN VERTEBRATES. ................................................................................ 18

FIGURE 2-1 SUFU IS DIFFERENTIALLY EXPRESSED IN THE GROWTH PLATE. .................................... 49

FIGURE 2-2 GENERATING CHONDROCYTE-SPECIFIC KNOCKOUT OF SUFU. ..................................... 50

FIGURE 2-3 SKELETAL PHENOTYPE ANALYSIS OF COL2A1-CRE; SUFUF/F

. ...................................... 52

FIGURE 2-4 SUFU NEGATIVELY REGULATES GROWTH PLATE CHONDROCYTE DIFFERENTIATION. ... 54

FIGURE 2-5 IN SITU HYBRIDIZATION ANALYSIS OF PTCH1 AND COL10A1 EXPRESSION IN VARIOUS

GENOTYPES. ............................................................................................................................ 56

FIGURE 2-6 KIF7 INACTIVATION RESULTED IN OPPOSITE EFFECTS ON CHONDROCYTE

PROLIFERATION AND DIFFERENTIATION TO THOSE OF SUFU INACTIVATION. ........................... 57

FIGURE 2-7 INCREASED LEVEL OF SUFU IN KIF7-DEIFICENT CHONDROCYTES. ............................... 59

FIGURE 2-8 INCREASED LEVEL OF SUFU-GLI COMPLEXES IN KIF7-DEIFICENT CHONDROCYTES. .... 60

FIGURE 2-9 A DUAL FUNCTION FOR KIF7 IN HH SIGNALING MODULATION. .................................... 62

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FIGURE 2-10 PROPOSED MODEL FOR HOW SUFU AND KIF7 REGULATE HH SIGNALING IN GROWTH

PLATE CHONDROCYTES. .......................................................................................................... 64

FIGURE 3-1 SUFU IS REQUIRED FOR THE NEGATIVE EFFECT OF PTHLH ON CHONDROCYTE

DIFFERENTIATION. .................................................................................................................. 87

FIGURE 3-2 KIF7 IS NOT REQUIRED FOR THE NEGATIVE EFFECT OF PTHLH ON CHONDROCYTE

DIFFERENTIATION. .................................................................................................................. 89

FIGURE 3-3 PTHLH INCREASES SUFU PROTEIN LEVEL. .................................................................... 90

FIGURE 3-4 PTHLH SIGNALING ACTIVATION PROMOTES GLI2 AND GLI3 PROTEIN PROCESSING VIA A

SUFU-MEDIATED MANNER. ..................................................................................................... 91

FIGURE 3-5 PROPOSED MODEL FOR HOW PTHLH REGULATES GLI TRANSCRIPTIONAL FACTORS

THROUGH A SUFU-MEDIATED MECHANISM. ............................................................................ 93

FIGURE 4-1 PROPOSED MODEL BY WHICH SUFU PROTEIN STABILITY SERVES AS A COMMON

MECHANISM THAT REGULATES GLI-MEDIATED TRANSCRIPTION. .......................................... 110

FIGURE 4-2 PROPOSED MECHANISM BY WHICH PTHLH AND IHH SIGNALING COORDINATE SUFU

EXPRESSION IN THE GROWTH PLATE. ..................................................................................... 111

FIGURE A- 1 THE PRIMARY CILIA ARE REQUIRED FOR NORMAL EMBRYONIC SKELETAL

DEVELOPMENT. ..................................................................................................................... 132

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FIGURE A- 2 ASYMMETRICAL HYPERTROPHY PHENOTYPE IN IFT88ORPK/ ORPK

MICE IS NOT SEEN AT

E18.5. ................................................................................................................................... 133

FIGURE A- 3 THE ROLE OF PRIMARY CILIA IN CHONDROCYTE HH LIGAND RESPONSIVENESS........ 134

FIGURE A- 4 THE ROLE OF PRIMARY CILIA IN MEDIATING PTHLH SIGNALING IN CHONDROCYTE

DIFFERENTIATION. ................................................................................................................ 135

FIGURE A- 5 INCREASED SUFU CILIARY LOCALIZATION IN PTHLH-TREATED WILD-TYPE

CHONDROCYTES.................................................................................................................... 136

FIGURE A- 6 A PROPOSED MODEL FOR CILIA-MEDIATED REGULATION OF GLI ACTIVITY. ............. 138

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Chapter 1 Introduction

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1.1 Summary

The work presented in the subsequent chapters is aimed at investigating Hh regulators,

Sufu, Kif7 and the primary cilium, in mediating Hh signaling as well as Pthlh signaling during

growth plate chondrocyte development. I examined the role of Sufu and Kif7 in regulating Hh

signaling in developing growth plate, and also proposed a model by which Sufu and Kif7 exert

distinct and overlapping functions in Hh pathway regulation. Furthermore, I examined how

these Hh regulators mediate the effects of Pthlh in chondrocytes. By dissecting the roles that

Sufu, Kif7 and the primary cilium play in regulating Hh and Pthlh signaling in the growth plate

chondrocytes. I provided insight into the mechanism by which Hh signaling is regulated and

highlighted Sufu as a common link between Ihh and Pthlh signaling in growth plate chondrocyte

development.

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1.2 Overview of growth plate development

Endochondral bone growth, which is the process of the cartilaginous skeletal template

progressively being replaced by bone, is precisely regulated by a number of signaling pathways

during development. Chondrocyte proliferation and differentiation are tightly controlled, and

dysregulation of these processes causes chondrodysplasias (generalized skeletal malformation

diseases), cartilaginous tumors, and osteoarthritis, a common degenerative joint disease. At the

onset of endochondral bone formation, mesenchymal cells condense and form a template of the

future skeletal element. This cartilage template is surrounded by the perichondrium, which is a

thin layer of flattened fibroblastic cells. Centrally, cells differentiate to chondrocytes and

proliferate and then mature from resting/low-proliferating chondrocytes into high-proliferating

cells that typically arrange in columns. The proliferating chondrocytes then undergo

differentiation successively into prehypertrophic and eventually hypertrophic chondrocytes. In

parallel, cells in the perichondrium adjacent to the hypertrophic region differentiate into

osteoblasts, which form the bone collar. The hypertrophic chondrocytes undergo programmed

cell death, and osteoblasts invade along with blood vessels and osteoclasts, leading to the

replacement of the cartilage template with trabecular bone (Kronenberg, 2003; Olsen et al., 2000;

Provot and Schipani, 2005). This process is illustrated in Figure 1-1. These layers of

chondrocytes proceeding in a staggered manner through steps of hypertrophic differentiation

constitute the growth plate, which is responsible for the longitudinal growth of the bones during

embryonic development and in the postnatal long bones.

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1.3 Indian hedgehog (Ihh) and Parathyroid hormone-like hormone

(Pthlh) signaling in growth plate development

1.3.1 Ihh/Pthlh feedback loop

A feedback loop formed by Ihh and Pthlh signaling functions to regulate the pace of

growth plate chondrocyte differentiation (Karp et al., 2000; Kobayashi et al., 2002; Lanske et

al., 1996; Minina et al., 2001). Ihh, which is expressed by prehypertrophic chondrocytes,

stimulates Pthlh expression in the articular region of the growth plate. Pthlh, which is a secreted

protein that acts on PTH/Pthlh receptor (PTHR1) bearing chondrocytes to maintain cells in a

proliferative state, thereby delaying the production of Ihh by prehypertrophic chondrocytes

(Kronenberg, 2003; Long et al., 2001) (Fig.1-2A). The levels of Ihh and Pthlh signaling

synchronize and regulate the pool of proliferating chondrocytes and the pace of chondrocyte

hypertrophic differentiation, thus determining the distance from the joint to which chondrocyte

hypertrophic differentiation take place (Karaplis et al., 1994; Lanske et al., 1996; St-Jacques et

al., 1999; Vortkamp et al., 1996).

1.3.2 Pthlh-dependent and –independent effects of Ihh

Loss of Ihh results in severe dwarfism due to accelerated chondrocyte hypertrophic

differentiation, reduced chondrocyte proliferation, and absence of osteoblasts in the bone collar

(Chung et al., 2001; Lanske et al., 1996; Long et al., 2004; Long et al., 2001; Maeda et al., 2007;

Razzaque et al., 2005; St-Jacques et al., 1999). Activation of Ihh signaling in Pthlh or PTHR1

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mutant limbs does not result in a delay in chondrocyte hypertrophic differentiation, suggesting

that the inhibitory effect of Ihh on chondrocyte hypertrophic differentiation is Pthlh-dependent

(Fig.1-2A). However, Ihh mutant mice (Ihh-/-

) exhibit a more severe reduction in the size of the

bone compared with Pthlh (Pthlh-/-

) or PTHR1 (PTHR1-/-

) mutants, whereas expression of

PTHR1 in Ihh mutant chondrocytes delays the accelerated onset of hypertrophic differentiation,

but does not possess any effect on the proliferation defect observed in Ihh mutant mice (Karp et

al., 2000). The data reveal a Pthlh-independent function of Ihh in chondrocyte proliferating (St-

Jacques et al., 1999) (Fig.1-2A). Further studies reveal that Ihh stimulates chondrocyte

proliferation in part through a cyclin D1-mediated mechanism (Duman-Scheel et al., 2002; Long

et al., 2001). The absence of bone collar in the Ihh-deficient bones reveals a positive role of Ihh

in regulating osteoblasts differentiation in the perichondrium. Interestingly, Ihh-/-

mice do not

exhibit severe defects in intramembranous bone formation (St-Jacques et al., 1999), which is

another essential process of mammalian skeletal development that contains the direct conversion

of mesenchymal tissue into bone. During later stages of osteoblast differentiation, inactivation of

Ihh signaling does not result in defects in the osteoblast differentiation process. The data suggest

that Ihh plays a role in initiating the differentiation of osteoblasts during endochondral skeletal

development (Fig.1-2A).

1.3.3 Actions of Pthlh in the growth plate

The binding of Pthlh to its receptor, PTHR1, stimulates G protein activation, which leads

to the production of cyclic AMP and activation of protein kinase A (PKA). The mechanism by

which cyclic AMP and PKA maintain chondrocytes in a proliferative state and delay

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chondrocyte hypertrophic differentiation is not completely understood. During mouse

embryogenesis, Sox9 plays an important role in regulating chondrocyte differentiation (de

Crombrugghe et al., 2001). Phosphorylation of Sox9 by Pthlh/PKA results in a more potent

activator function in upregulating target genes (Huang et al., 2001); thus, Sox9 phosphorylation

could contribute to the negative effect of Pthlh on chondrocyte hypertrophic differentiation. In

addition to its inhibitory role in regulating chondrocyte hypertrophic differentiation, Pthlh also

exhibits a stimulatory effect on chondrocyte proliferation. In contrast to the PKA-mediated

action of Pthlh on chondrocyte hypertrophic differentiation, its pro-proliferative effect is not

mimicked by PKA activation. Deletion of p57 results in a growth plate phenotype mimicking

what was observed in Pthlh-null mice, including increased chondrocyte proliferation and delayed

chondrocyte hypertrophic differentiation. Double knockout of Pthlh and p57 attenuates the

growth plate defects in Pthlh-null mice (MacLean et al., 2004). The data suggest that the

stimulatory effect of Pthlh on chondrocyte proliferation is mediated in part through decreasing

p57 expression in the growth plate (MacLean et al., 2004).

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1.4 Ihh and Pthlh signaling pathways regulate Gli transcription factors

Ihh, a member of the Hedgehog (Hh) family of signaling molecules, regulates Gli

transcriptional activity through binding to its receptor Patched 1 (Ptch1) and derepressing the

signal transducer Smoothened (Smo). Three Gli zinc finger proteins Gli1, Gli2 and Gli3 (glioma-

associated oncogene family member 1, 2 and 3) are transcription factors mediating Hh pathway

in mammalian cells (Jiang & Hui, 2008). Analysis of genetically modified mice show that Gli2

and Gli3 act as essential mediators in Hh signaling, whereas Gli1 is dispensable for embryonic

development and acts as a secondary mediator of Hh signaling transduction (Bai et al., 2004;

Ding et al., 1998; Mo et al., 1997; Motoyama et al., 1998; Park et al., 2000). Although all three

Gli proteins can function as activators of the pathway, Gli2 and Gli3 are the major transcriptional

activator and repressor of the mammalian Hh signaling, respectively (Bai et al., 2004; Buttitta et

al., 2003; McDermott et al., 2005; Motoyama et al., 1998).

In the absence of Hh ligand, Gli3 is phosphorylated sequentially by PKA, glycogen

synthase kinase 3ß (GSK3ß) and casein kinase 1 (CK1) and targeted to ubiquitin/proteasome-

mediated proteolysis to generate a C-terminally truncated transcriptional repressor (Gli3R)

(Tempe et al., 2006; Wang et al., 2000; Wang and Li, 2006). Gli2 is also phosphorylated by

these kinases, although in the case of processing is inefficient and leads to mostly degradation by

the proteasome (Pan et al., 2006). The specific domain in the C-terminus has been suggested to

be responsible for the differential processing and degradation of Gli2 and Gli3 (Pan and Wang,

2007). Hh signaling activation blocks the proteolytic cleavage of Gli2 and promotes the activator

function of Gli2 and Gli3. However the mechanism is undefined.

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Data from mutant mouse analyses suggest that Gli2 and Gli3 are involved in Ihh-

dependent growth plate chondrocyte development. Ihh-null mice show reduced chondrocyte

proliferation, increased zone of hypertrophic chondrocytes and lack of ossification in

endochondral bones (St-Jacques et al., 1999). Similarly, Gli2 knockout mice display expanded

hypertrophic zone and reduced bone formation, suggesting that Gli2 functions as an activator in

regulating Hh signaling in chondrocytes and reduced Gli2 function is in part responsible for the

Ihh-null phenotype (Miao et al., 2004). Genetic studies in mice show that Gli3 functions as a

major repressor regulating Hh signaling in growth plate chondrocytes. Loss of function of Gli3

rescues the growth plate developmental defects in Ihh-null mice, which supports the notion that

Gli3 acts as a negative regulator of Hh-mediated signaling in chondrocytes and Ihh possess

inhibitory effect on the repressor function of Gli3 in this process (Koziel et al., 2005).

Besides the Hh-mediated regulation, the Gli transcription factors may also be regulated

through an Hh/Smo-independent mechanism. Studies of explants lacking the Gli genes suggest

that Gli3, but not Gli2, is involved in the inhibitory effect of Pthlh on growth plate chondrocyte

hypertrophic differentiation. Pthlh signaling activation inhibits Gli-mediated transcription

through processing Gli3 via a PKA-dependent manner (Mau et al., 2007; Miao et al., 2004;

Tukachinsky et al., 2010; Tuson et al., 2011; Vortkamp et al., 1996; Zeng et al., 2010 (Fig1-2B).

These results suggest another level of feedback control between Ihh and Pthlh signaling and raise

the possibility that the Gli transcription factors act as integrators of multiple signaling pathways

during growth plate chondrocyte differentiation. However, our knowledge of the regulation of

Gli-mediated pathway activity is still limited.

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1.5 Regulators in Hedgehog (Hh) signaling pathway: Sufu and Kif7

In mammalian Hh signaling, Suppressor of fused (Sufu) and Kinesin family member 7

(Kif7) are two evolutionarily conserved regulators of Gli transcription factors. Deletion of Sufu

results in embryonic lethality at E9.5 with severe ectopic Hh signaling activation, indicating Sufu

functions as a major repressor of mammalian Hh pathway (Cooper et al., 2005; Svard et al.,

2006). Western analysis revealed a severe reduction in the levels of Gli2 and full length Gli3 as

well as a lack of Gli3 repressor in Sufu-/-

embryos (Humke et al.; Wang et al., 2010), suggesting a

role of Sufu in controlling Gli transcriptional repressor processing. However, the mechanism of

action of Sufu remains controversial. Sufu interacts directly with all three Gli proteins and

sequesters them in the cytoplasm in the absence of Hh ligand, which prevents Hh pathway

activation (Barnfield et al., 2005; Ding et al., 1999; Kogerman et al., 1999; Murone et al., 2000).

Sufu acts to recruit a co-repressor complex to inhibit Gli activator function (Cheng and Bishop,

2002) and promotes Gli3R formation through interacting with GSK3ß (Kise et al., 2009). Recent

studies further reveal a role for Sufu in maximal Gli-dependent transcriptional activation,

suggesting that Sufu also acts positively in Hh signaling (Chen et al., 2009). Hh signaling

activation promotes Sufu protein turnover via the ubiquitin-proteasome system (Yue et al.,

2009). PKA and GSK3ß phosphorylate Sufu at Ser-346 and Ser-342, respectively, and

phosphorylation stabilizes Sufu against Hh signaling-induced degradation (Chen et al., 2011).

Taken together, the data suggest that Sufu plays an important role in regulating Gli activities, and

Sufu itself is tightly regulated to achieve a gradient level of Hh signaling activity during

development.

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In contrast, less is known about the action of Kif7. Loss of Kif7 results in perinatal death

and phenotypes, such as polydactyly, mimicking Gli3 knockout mice (Chen et al., 2009; Cheung

et al., 2009; Hui and Joyner, 1993). Kif7 also interacts with all three Gli proteins and elevated

Gli2 and reduced Gli3 levels are found in Kif7-null mice, suggesting that it acts as a negative

regulator of the mammalian Hh pathway. Kif7-/-

embryos exhibit defects in floor plate

development, which depends on maximal level of Hh pathway activation, indicating a potential

positive role of Kif7 in Hh signaling (Cheung et al., 2009). The mechanism by which Sufu and

Kif7 execute dual functions and/or whether they possess cooperative regulatory functions is not

known.

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1.6 The primary cilium in Hh signaling

In vertebrates, the primary cilium, which is a non-motile microtubule-based organelle,

serves as a focal point in Hh signaling regulation (Corbit et al., 2005; Goetz and Anderson, 2010;

Huangfu et al., 2003). The formation and maintenance of the primary cilium depend on

intraflagellar transport (IFT) of protein complexes driven by kinesin and dynein motors for

anterograde and retrograde IFT movements, respectively. Mutation in proteins involved in IFT,

such as IFT88, IFT172, and the motor protein Kif3a, prevents functional cilia formation and

attenuates Hh signaling (Huangfu et al., 2003). To date, many components of the Hh pathway are

found to be localized to the cilia either in the absence of Hh ligand or during pathway activation

(Corbit et al., 2005; Haycraft et al., 2005; Huangfu and Anderson, 2005; Liu et al., 2005). Ptch1

is localized to the primary cilia where it prevents the ciliary localization of Smo in the absence of

Hh ligand. Binding of Hh ligand to Ptch1 induces its export from the cilia, which allows the

ciliary translocation of Smo. Through an unknown mechanism, ciliary localization of Smo relays

Hh signals to the cytoplasm, which leads to Gli-mediated transcription activation (Rohatgi et al.,

2007) (Fig. 1-3).

Hh pathway activation in the absence of Sufu is independent of cilia (Chen et al., 2009;

Jia et al., 2009), suggesting that Smo may activate Gli proteins at the cilia by inhibiting Sufu.

PKA downregulates Smo-mediated Hh signaling activation via a Sufu-dependent mechanism

(Chen et al., 2009; Svard et al., 2006; Wu et al., 2004), suggesting that Smo and PKA both

modulate Hh signaling thorough Sufu. Therefore, a model by which Hh signaling activates Gli

protein at the cilia through relieving the inhibitory function of Sufu, resulting in Gli-mediated

12

transcriptional activation was proposed. This model is supported by recent studies carried out in

cultured fibroblasts, showing that Smo plays a positive role in dissociation of inhibitory Sufu-Gli

complexes at the primary cilium upon Hh pathway activation. Furthermore, Gli2 and Gli3 are

required to recruit Sufu to cilia, but Gli proteins can localize to cilia in the absence of Sufu

(Humke et al., 2010; Tukachinsky et al., 2010). However, how Hh signaling and/or other

signaling pathways regulate Sufu in the context of primary cilia is unknown. Mammalian Sufu

bears four conserved cAMP-dependent PKA recognition sites, and phosphorylation by PKA and

GSK3ß stabilizes Sufu and promotes its tendency to stay longer at the ciliary tip (Chen et al.,

2011) (Fig. 1-3). This stabilizing effect of PKA on Sufu is consistent with its inhibitory function

on Hh signaling (Dai et al., 1999; Tempe et al., 2006). A carboxy-terminal region of Sufu

interacts and recruits GSK3ß for efficient Gli3 processing (Kise et al., 2009). It is plausible that

Sufu, stabilized through phosphorylation by PKA and GSK3ß, forms a complex with Gli2/3 and

is transported into the primary cilium where further modification takes place, allowing formation

of the truncated transcriptional repressors via proteolytic processing in the cytoplasm (Fig.1-3).

We do not yet know how the integrity of the Sufu-Gli complex is maintained or how the

phosphorylation state of Sufu and/or Gli regulates this complex. We do not yet know how Hh

signaling promotes Sufu-Gli complexes dissociation or whether this process can take place

outside of the primary cilium. Additionally, it will be interesting to know if Kif7, which shows

dynamic ciliary localization (Liem et al., 2009), plays a regulatory role in the formation and/or

dissociation of Sufu-Gli complexes in the primary cilium. Furthermore, during growth plate

chondrocyte differentiation, Pthlh exerts regulatory functions on Gli proteins as well as Gli-

mediated transcriptional activity, and it functions mainly through PKA activation. Thus, Sufu

regulation may serve as a common and important regulatory mechanism between Ihh and Pthlh

13

signaling during growth plate chondrocyte development.

14

1.7 Hypothesis & Specific aims

Proper regulation of Hh and Pthlh signaling is crucial in chondrogenesis. Hh signaling

mediators, such as Sufu, Kif7, and primary cilium, may play important roles in controlling the

process of chondrocyte differentiation. I hypothesize that Sufu, Kif7, and primary cilium play

distinct and overlapping roles in regulating Hh signaling activity during chondrocyte

development.

There are three specific aims set out for this work:

Aim 1: What are the regulatory roles of Sufu and Kif7 in Hh signaling during growth plate

chondrocyte development?

Aim 2: What is the role of Kif7 in the formation and/or dissociation of Sufu-Gli complexes?

Aim 3: What are the potential roles of Sufu, Kif7, and primary cilium in Pthlh action during

chondrocyte development?

15

1.8 Figures

Figure 1-1 Schematic representation of endochondral ossification.

Figure 1-1. Schematic representation of endochondral ossification. (A) Mesenchymal cells

condense to form the model of the bone. (B-C) Centrally, cells differentiate into chondrocytes

and start to proliferate and undergo several steps of differentiation into hypertrophic

chondrocytes. (D-E) Hypertrophic chondrocytes are postmitotic and express Col10a1.

Osteoblasts invade along with blood vessels and osteoclasts to replace the cartilaginous template

with trabecular bone. (F) Perichondrial cells adjacent to the hypertrophic chondrocytes become

osteoblasts, which forms the bone collar. Postnatally, secondary ossification center forms at each

end of the long bone.

16

Figure 1-2 Ihh/Pthlh signaling regulation in the growth plate.

17

Figure 1-2. Ihh/Pthlh signaling regulation in the growth plate. (A) Growth plate chondrocytes

undergo a coordinated differentiation process, ending in chondrocyte hypertrophy and apoptosis.

1) Ihh, which is expressed by prehypertrophic chondrocytes, stimulates Pthlh expression at the

periarticular region that functions to inhibit chondrocyte hypertrophic differentiation and Ihh

expression (illustrated by 4.). 2) Ihh positively regulates chondrocyte proliferation (circled P) via

a Pthlh-independent manner. 3) Ihh initiates osteoblast differentiation at the perichondrium

adjacent to its expressing region. 4) Pthlh inhibits chondrocyte hypertrophic differentiation

mainly through PKA activation. 5) Pthlh possesses stimulatory effect on chondrocyte

proliferation. Solid lines show the regulation of Pthlh, and dashed lines show the regulation of

Ihh. (B) Both Gli2 and Gli3 are involved in the regulation of chondrocyte hypertrophic

differentiation. Pthlh negatively regulates this differentiation process through processing Gli3FL

into its truncated form (Gli3R) via a PKA-dependent mechanism.

18

Figure 1-3 Hh signaling in vertebrates.

Figure 1-3. Hh signaling in vertebrates. In the absence of the Hh ligand, Ptch1 localizes to the

primary cilium and prevents Smo ciliary accumulation. Phosphorylation by PKA and GSK3ß

stabilizes Sufu and promotes its tendency to stay longer at the ciliary tip. Sufu-Gli complexes

traffic through the cilium at low level and promote Gli repressor (GliR) formation. Hh ligand

binding to Ptch1 leads to the translocation of Smo into the cilium. Smo-mediated dissociation of

Smo-Gli complexes results in Gli transcriptional activation.

19

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25

Chapter 2 Kif7 promotes Hh signaling in growth plate

chondrocytes by restricting the inhibitory function of Sufu

Shu-Hsuan C. Hsu, Xiaoyun Zhang, Chunying Yu, Zhu Juan Li, Jay S. Wunder, Chi-Chung Hui,

and Benjamin A. Alman

All experiments and analysis described in this chapter were conducted by Shu-Hsuan C. Hsu

with the exception of the western and in situ analysis by Xiaoyun Zhang and Chunying Yu. This

study has been published in Development (Hsu et al., 2011).

26

2.1 Summary

Proper regulation of Indian hedgehog (Ihh) signaling is vital for chondrocyte proliferation

and differentiation in the growth plate. Its dysregulation causes skeletal dysplasia, osteoarthritis,

or cartilaginous neoplasia. Here, I show that Suppressor of fused (Sufu) and Kif7 are essential

regulators of Ihh signaling. While Sufu acts as a negative regulator of Gli transcription factors,

Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in the turnover

of Sufu and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving

the dosage of Sufu restores normal Hedgehog pathway activity and chondrocyte development in

Kif7 null mice demonstrating that the positive role of Kif7 is to restrict the inhibitory activity of

Sufu. Furthermore, Kif7 also inhibits Gli transcriptional activity in the chondrocytes when Sufu

function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both

Sufu-dependent and -independent mechanisms.

27

2.2 Introduction

The precise regulation of chondrocyte proliferation and differentiation is critical for

normal bone growth. During endochondral bone development, growth plate chondrocyte

differentiation is governed by the spatial and temporal regulation of a number of signaling

pathways. Dysregulation of these processes during development is responsible for skeletal

dysplasias, which are characterized by short stature (Karsenty et al., 2009). Inhibition of

differentiation of growth plate chondrocytes can cause cartilaginous tumors (Bovee et al., 2010)

and aberrant activation of signaling pathways normally involved in the regulation of growth plate

chondrocytes is associated with osteoarthritis, a common degenerative joint disease (Lin et al.,

2009). Therefore, abnormal chondrocyte proliferation and differentiation have profound

negative effects on overall health.

Ihh, a member of the Hedgehog (Hh) family of signaling molecules, regulates the

transcriptional activity of Gli proteins through binding to its receptor Patched1 (Ptch1) and

derepression of the signaling receptor Smoothened (Smo). Three Gli zinc finger proteins (Gli1-3)

are transcription factors mediating Hh signaling in mammalian cells (Jiang & Hui, 2008). In

mice, Gli2 and Gli3 are essential genes, whereas Gli1 is dispensable for embryonic development

and encodes a secondary mediator of Hh signaling. Gli2 and Gli3 are the major transcriptional

activator and repressor of the mammalian Hh pathway, respectively, though all three Gli proteins

can activate the expression of Hh target genes, such as Ptch1 and Gli1 itself. Gli3 is processed

efficiently by the proteasome into a C-terminally truncated transcriptional repressor. Through ill-

defined mechanisms, Hh signaling blocks the proteolytic cleavage of Gli3 and promotes the

28

transcriptional activator function of Gli2 and Gli3. Mutant mouse analysis indicates that Gli2 and

Gli3 are involved in Ihh-dependent chondrocyte development. Mice lacking Ihh are

characterized by reduced chondrocyte proliferation, an expanded hypertrophic zone in the

growth plate, and lack of ossification in endochondral bones (St-Jacques et al., 1999). Similar to

Ihh knockout mice, Gli2 knockout mice show an expanded hypertrophic zone and reduced bone

formation, suggesting that the Ihh mutant phenotype is in part due to a reduction of Gli2

activator function (Miao et al., 2004). Loss of Gli3 rescues the chondrocyte proliferation and

differentiation defects in Ihh mutant mice indicating that a major action of Ihh is to limit the

repressor function of Gli3 in growth plate chondrocytes (Koziel et al., 2005). These observations

indicate that Ihh-dependent regulation of Gli2 and Gli3 plays a critical role in chondrocyte

differentiation.

In mammalian Hh signaling, Sufu and Kif7 are two evolutionarily conserved regulators

of Gli transcription factors (Wilson et al., 2009). In mice, Sufu is a major negative regulator of

Hh signaling and inactivation of Sufu leads to embryonic lethality at E9.5 with severe ectopic Hh

pathway activation similar to those observed in Ptch1-null embryos (Cooper et al., 2005; Svard

et al., 2006). Sufu forms complexes with all three Gli proteins and inhibits their transcriptional

activity (Barnfield et al., 2005; Ding et al., 1999). Recent studies in cultured fibroblasts have led

to the suggestion that Hh signaling promotes the nuclear translocation and transcriptional activity

of Gli2 and Gli3 through dissociation of cytoplasmic Sufu-Gli complexes (Humke et al., 2010;

Tukachinsky et al., 2010). In addition, Sufu-/-

cells exhibit a drastic reduction in the levels of full

length Gli2 and Gli3 as well as a lack of Gli3 repressor suggesting that it also plays a critical role

in the stabilization of Gli activators and the formation of Gli3 repressor (Humke et al., 2010;

Wang et al., 2010). In contrast, less is known about the action of Kif7, which is a kinesin motor

29

protein recently shown to play regulatory roles in mammalian Hh signaling. Kif7-null mice die at

birth and exhibit a phenotype (Chen et al., 2009; Cheung et al., 2009) similar to that of Gli3-null

mice (Hui and Joyner, 1993). Kif7-null embryos show mild ectopic Hh pathway activation with

ectopic formation of ventral neurons in the spinal cord as well as elevated Gli2 and reduced Gli3

levels, suggesting that it acts negatively in Hh signaling. Interestingly, Kif7 also functions

positively in controlling Hh pathway activity (Cheung et al., 2009; Liem et al., 2009). For

example, floor-plate development, which is induced by maximal level of Hh pathway activity, is

compromised in the absence of Kif7 function (Adolphe et al., 2006; Cheung et al., 2009; Endoh-

Yamagami et al., 2009). How Kif7 acts both negatively and positively in mammalian Hh

signaling is not understood and whether Kif7 possesses cooperative regulatory function with

Sufu is unclear.

The primary cilium acts as a focal point in the processing of Hh signaling (Corbit et al.,

2005; Goetz and Anderson, 2010; Huangfu et al., 2003). Recent studies have suggested that,

when the Hh pathway is activated, Smo promotes the dissociation of inhibitory Sufu-Gli

complexes at the primary cilium (Tukachinsky et al., 2010). Kif7 has been shown to translocate

to the tip of primary cilium in cultured fibroblasts upon pathway stimulation (Liem et al., 2009).

However, it is unknown whether Kif7 plays a regulatory role in the formation and/or dissociation

of Sufu-Gli complexes in the primary cilium. In this study, I explored the roles of Sufu and Kif7

in Ihh-dependent chondrocyte development using genetically modified mice. Our results indicate

that while Sufu is a major negative regulator of Hh pathway activity, Kif7 plays dual roles in the

control of chondrocyte development. Intriguingly, Kif7 is localized to the ciliary tip of

proliferating chondrocytes in vivo and appears to exclude Sufu-Gli complexes from the primary

cilium. I speculate that Kif7 functions positively in Hh signaling to promote Smo-induced

30

dissociation of Sufu-Gli complexes at the primary cilium.

31

2.3 Results

2.3.1 Sufu is differentially expressed in the growth plate and is required for normal

skeletal development

To determine if Sufu might play a role in growth plate chondrocyte function, Sufu

expression was examined in fetal limb cartilage. qRT-PCR analysis on micro-dissected sections

of the growth plate and in situ hybridization were used to localize its RNA expression. Sufu is

highly expressed in articular and resting chondrocytes (also known as reserve chondrocytes; the

early stages of chondrocyte differentiation), while its expression is downregulated when cells

undergo differentiation to hypertrophic chondrocytes (Fig. 2-1A, D). Immunohistochemical

staining of fetal limbs revealed high level of Sufu protein expression in resting and proliferating

chondrocytes, and that fewer than 10% of the prehypertrophic and hypertrophic chondrocytes are

Sufu-positive (Fig. 2-1C, E). The pattern of Sufu expression is similar to that of Pthlh (an Hh

regulated gene in the growth plate), but in contrast, Ihh expression is highest in the

prehypertrophic chondrocytes (Fig. 2-1B). Thus, Sufu is differentially expressed in the growth

plate, with its highest level in early stages of chondrocyte differentiation.

To bypass the embryonic lethality of Sufu-null mice at E9.5 (Cooper et al., 2005; Svard et

al., 2006), I generated chondrocyte-specific knockout mice (Col2a1-Cre; Sufuf/f

) to investigate

the role of Sufu in growth plate chondrocyte development. Immunohistochemical staining and

western analysis demonstrated efficient deletion of Sufu in the growth plate of Col2a1-Cre;

Sufuf/f

mice (Fig. 2-2A-C). Chondrocyte-specific knockout of Sufu in mice resulted in perinatal

death with a few exceptions that survived for 10 days. Body length and weight measurements

32

demonstrated a ~24% and ~50% reduction in the mutants, respectively, when compared with

wild-type littermates (Fig. 2-2D-F). Alcian blue/Alizarin red staining showed a significant

reduction of bone ossification in Col2a1-Cre; Sufuf/f

mice (Fig. 2-3A, D). Histological analysis

revealed delayed formation of secondary ossification centers, expansion of proliferating zone as

well as reduction of hypertrophic zone in the Sufu-deficient tibial and vertebral growth plates

(Fig. 2-3B). By Ki67 and Phospho-H3 immunostaining, a higher percentage of proliferating cells

was found in the mutant growth plate (Fig. 2-3E, F; Fig. 2-6J). In contrast, immunostaining of

active Caspase-3 and TUNEL analysis did not reveal a significant difference of apoptosis in the

mutants (Fig. 2-3G). These data indicate that Sufu is required for normal endochondral skeletal

development, where it regulates growth plate chondrocyte proliferation and differentiation.

2.3.2 Sufu acts as a negative regulator of Hh signaling during chondrocyte

differentiation

Ptch1 inhibits Smo in the absence of Hh signals and acts as a negative regulator of Hh

signaling. Chondrocyte-specific knockout of Ptch1 (Col2a1-Cre; Ptch1f/f

) leads to elevated Hh

pathway activity and results in a phenotype similar to those observed in Sufu mutants, including

an expansion of proliferative zone and a reduction of hypertrophic zone (Mak et al., 2008) (Fig.

2-3C; Fig. 2-4A-C). I found that the Sufu knockout phenotype is consistently milder than the

Ptch1 knockout phenotype (Fig. 2-4A-C). To determine whether knockout of Sufu also results in

Hh pathway activation in the chondrocytes, I performed qRT-PCR analysis. Knockout of Ptch1

leads to increased expression of Hh target genes, such as Gli1, Ptch1, and Hhip1 (Fig. 2-4D).

Interestingly, I detected up-regulation of Ptch1 and Hhip1, but not Gli1, in Sufu-deficient

33

chondrocytes (Fig. 2-4D). Analysis by in situ hybridization revealed Ptch1 transcripts in the

proliferating chondrocytes. In wild-type mice, cells adjacent to Ihh-producing prehypertrophic

chondrocytes show highest levels of Ptch1 transcripts and Ptch1 expression decreases toward the

end of the bone. In addition to proliferative zone, Sufu-deficient mice exhibited higher levels of

Ptch1 transcripts also in the resting chondrocytes (Fig. 2-5A, C). Furthermore, loss of Sufu

resulted in a reduction of the hypertrophic zone as verified by in situ hybridization analysis for

collagen X expression (Fig. 2-5E, G). Western analysis revealed an increase (2-fold) in the level

of Gli2 protein as well as an increase (1.8-fold) in the ratio of full-length versus repressor form

of Gli3 (Gli3FL:Gli3R) in Sufu-deficient chondrocytes (Fig. 2-4E). Together, these data suggest

that Sufu, like Ptch1, functions as a negative regulator of Hh signaling in chondrocytes but its

inactivation only leads to partial pathway activation.

2.3.3 Loss of Kif7 in growth plate chondrocytes results in reduced Hh pathway

activity

As Sufu inactivation did not lead to a phenotype as severe as that of Ptch1 inactivation, I

reasoned that other pathway components might cooperate with Sufu in regulating Hh signaling.

One potential candidate is Kif7, which was recently shown to be a negative regulator of Gli

transcription factors (Cheung et al., 2009). Kif7 is expressed at high levels in the articular/resting

chondrocytes and its expression is drastically down-regulated in proliferating, prehypertrophic,

and hypertrophic chondrocytes demonstrated by qRT-PCR on micro-dissected sections of the

growth plate, in situ hybridization, and immunostaining (Fig. 2-6A-C). To investigate whether it

functions as a regulator of Ihh signaling, I examined the chondrocyte phenotype of Kif7-null

34

mice (Cheung et al., 2009). Kif7-/-

mice die at birth and as such, the growth plates of E16.5 mice

were analyzed. Contrary to that observed in chondrocyte-specific Sufu knockout mice, I found

that Kif7-null mice exhibit a reduction in the size of the proliferative zone and an expansion of

hypertrophic zone (Fig. 2-6D, F, G). To rule out the possibility that the growth plate phenotype is

due to secondary effects caused by Kif7 inactivation in other cells types, I generated

chondrocyte-specific Kif7 knockout mice (Col2a1-Cre; Kif7f/f

). Col2a1-Cre; Kif7f/f

mice appear

normal and do not exhibit any gross defects (Fig. 2-6E). No obvious phenotypic difference was

found in tibial growth plates of P10 Kif7-deficient mice compared to their wild-type counterparts

(Fig. 2-6H). However, histological and in situ hybridization analyses of E16.5 Col2a1-Cre;

Kif7f/f

tibia revealed a reduction of proliferative zone and an expansion of hypertrophic zone (Fig.

2-6F, G; Fig. 2-5E, F) similar to those observed in Kif7-/-

mice. Furthermore, the growth plates

of Kif7-/-

and Col2a1-Cre; Kif7f/f

mice showed a reduction in cell proliferation demonstrated by

Ki67 and Phospho-H3 immunostaining (Fig. 2-6I, J). These results indicate that the effects of

Kif7 inactivation on chondrocyte proliferation and differentiation are opposite to those of Sufu

inactivation, and suggest that Kif7 acts as a positive regulator of Ihh signaling. Consistent with

this notion, Kif7 inactivation leads to a down-regulation of Hh target genes, Gli1 and Ptch1, as

revealed by qRT-PCR analysis (Fig. 2-9D), and reduced Ptch1 mRNA expression in the

proliferating region as illustrated by in situ hybridization analysis (Fig. 2-5A, B). Therefore, in

contrast to its role as a negative regulator of Hh signaling in early mouse embryos (Cheung et al.,

2009), Kif7 functions positively in Ihh signaling during growth plate chondrocyte development.

2.3.4 Sufu-Gli complexes are localized to the ciliary tip in the absence of Kif7

35

To investigate whether loss of Kif7 affects the functional activity of Sufu in chondrocytes

and vice versa, I first examined their expression in Kif7 and Sufu mutant mice. While no

difference in Kif7 protein or RNA levels was detected in Sufu-deficient chondrocytes (Fig. 2-7A,

B), I found a substantial increase of Sufu protein levels in Kif7-deficient chondrocytes (Fig. 2-

7C). To determine if this is caused by altered RNA expression or protein stability, I examined

Sufu transcript levels using qRT-PCR (Fig. 2-7D) and Sufu protein levels using pulse chase

experiments in the presence of cycloheximide (Fig. 2-7E, F). These experiments demonstrated

that elevated Sufu protein levels are due to increased stability in Kif7-deficient chondrocytes.

Furthermore, treatment with MG132, an inhibitor of proteasome degradation, resulted in a higher

level of Sufu protein in wild-type, but not in Kif7-deficient chondrocytes, indicating that Sufu

protein is rapidly degraded by the proteasome in wild-type chondrocytes (Fig. 2-7G, H).

Together, these data suggest that Kif7 plays a role in the turnover of Sufu in wild-type

chondrocytes.

The observations that Smo activation promotes the dissociation of Sufu-Gli complexes at

the primary cilium prompted us to investigate the ciliary localization of Sufu and Kif7 in the

growth plate chondrocytes and to examine whether Kif7 influences Sufu-Gli complexes in their

primary cilia. In Gli2-/-

;Gli3-/-

embryonic fibroblasts, where all Gli proteins are absent, Sufu is

not detected in the cilia even upon Shh stimulation demonstrating that Gli proteins are necessary

to recruit Sufu to the cilia (Tukachinsky et al., 2010; Zeng et al., 2010). Thus, Sufu

immunostaining could serve as an indicator of Sufu-Gli complexes at the primary cilium. In

proliferating chondrocytes (where the Hh pathway is active), Kif7 was mostly localized to the

ciliary tip, whereas Sufu was found at the basal bodies in ~50% of these cells but was rarely

detected at the ciliary tip (Fig. 2-8A, B). Similarly, Gli2 and Gli3 staining were rarely found in

36

the primary cilium of these chondrocytes (Fig. 2-8A, B). Both Sufu and Kif7 signals are specific

since they are absent in Sufu-deficient and Kif7-deficient chondrocytes, respectively. Together,

these results indicate that there is very little Sufu-Gli2 or Sufu-Gli3 complexes present in the

primary cilium of proliferating wild-type chondrocytes.

Loss of Sufu has no apparent effects on the ciliary localization of Kif7 (Fig. 2-8A, C).

Consistent with previous studies done in cultured fibroblasts (Humke et al., 2010; Tukachinsky

et al., 2010), Gli2 and Gli3 could be detected at the ciliary tip in ~10% of the Sufu-deficient

chondrocytes (Fig. 2-8A, C), suggesting that Gli proteins can translocate to the cilia independent

of Sufu. Strikingly, in the absence of Kif7, ~20% and ~60% of proliferating chondrocytes

showed Gli2 and Gli3 staining at the ciliary tip, respectively (Fig. 2-8A, D). Consistent with the

notion that Sufu and Glis are transported to the cilia as a complex, Sufu is localized to the ciliary

tip of ~90% of Kif7-deficient chondrocytes (Fig. 2-8A, D). These observations indicate that both

Sufu-Gli2 and Sufu-Gli3 complexes accumulate in the primary cilium of Kif7-deficient

chondrocytes, and suggest that when the Hh pathway is activated in chondrocytes, Kif7 plays a

key role in excluding them from the primary cilia. I propose that Smo promotes the dissociation

of Sufu-Gli complexes in the primary cilium through the action of Kif7.

To determine whether Ihh signaling promotes the dissociation of Sufu-Gli complexes in

chondrocytes, primary chondrocyte cultures were treated with cyclopamine (Smo antagonist),

purmorphamine (Smo agonist) or Shh, and Sufu-Gli2 complexes were quantified using western

analysis of Gli2 followed by immunoprecipitation with Sufu-specific antibodies. There was a

similar level of Gli2 in all samples. While Gli2 levels were similar in the Sufu-

immunoprecipitate from control and cyclopamine-treated chondrocytes, there was a significant

reduction of Gli2 levels in those of purmorphamine- and Shh-treated chondrocytes (Fig. 2-8E,

37

F). These results indicate that, similar to those observed in cultured fibroblasts, Smo activation

also promotes the dissociation of Sufu-Gli2 complexes in the chondrocytes.

2.3.5 Removal of one copy of Sufu rescues the Kif7 mutant growth plate phenotype

To investigate whether increased Sufu protein levels contribute to the reduced Hh

pathway activity in Kif7-deficient chondrocytes, I generated Col2a1-Cre; Sufuf/+

; Kif7f/f

mice.

Strikingly, these mice develop a normal growth plate (Fig. 2-9A). The expansion of hypertrophic

zone and the reduction of proliferative zone observed in Col2a1-Cre; Kif7f/f

mice are almost

completely rescued by the simultaneous removal of one dose of Sufu (Fig. 2-9A-C). Importantly,

Gli1 and Ptch1 expression was also restored to near wild-type levels in Col2a1-Cre; Sufuf/+

;

Kif7f/f

chondrocytes (Fig. 2-9D). These data clearly indicate that the reduced Hh pathway activity

in Kif7-deficient chondrocytes is due to increased Sufu protein level and suggest that the positive

role of Kif7 in Ihh signaling during chondrocyte differentiation is in part through the down-

regulation of Sufu protein expression as well as the dissociation of Sufu-Gli complexes (Fig. 2-

10A, B).

2.3.6 Kif7 and Sufu share overlapping functions in Hh signaling during

chondrocyte development

The co-expression of Sufu and Kif7 in articular/resting chondrocytes prompted us to

analyze whether they possess additional overlapping functions in Ihh signaling and chondrocyte

38

development. I generated Col2a1-Cre; Sufuf/f

mice with additional deletion of one allele of Kif7

(Col2a1-Cre; Sufuf/f

; Kif7f/+

) as well as Sufu; Kif7 double knockout mice (Col2a1-Cre; Sufuf/f

;

Kif7f/f

). While deletion of one allele of Kif7 in Sufu-deficient growth plate resulted in a similar

phenotype as Col2a1-Cre;Sufuf/f

mice, Col2a1-Cre;Sufuf/f

;Kif7f/f

mice showed a more severe

phenotype (Fig. 2-9A-C; Fig. 2-5E, H), including further reduction of hypertrophic zone and

expansion of proliferative zone demonstrated by immunohistochemical staining and in situ

hybridization analyses, similar to those observed in Col2a1-Cre;Ptch1f/f

mice (Fig. 2-4A).

Importantly, I found that inactivation of both Sufu and Kif7 leads to augmented expression of all

three Hh target genes Ptch1 and Hhip1 as well as Gli1 (Fig. 2-9D). By in situ hybridization, I

found that Ptch1 expression is elevated in both the resting and proliferating regions of Sufu;Kif7-

deficient mice when compared with that of Sufu-deficient mice (Fig. 2-5A, C, D). Thus, in the

absence of Sufu, Kif7 functions instead as a negative regulator of the Hh pathway suppressing

the expression of Hh target genes. I examined the localization of Gli2 and Gli3 in the primary

cilium of Sufu; Kif7-deficient chondrocytes. Interestingly, neither Gli2 nor Gli3 is localized to

the ciliary tip of Sufu; Kif7-deficient chondrocytes suggesting that Gli proteins cannot be

processed in the cilium in the absence of both Sufu and Kif7 (data not shown). Together, these

observations indicate that both Sufu and Kif7 could contribute to the negative regulation of Gli

transcription factors and their concomitant deletion results in maximal Hh pathway activation,

similar to that observed in cells lacking Ptch1. Therefore, Kif7 possesses both positive and

negative roles in the regulation of Ihh signaling during chondrocyte development (Fig. 2-10A,

B).

39

2.4 Discussion

In this study, I demonstrated distinct and overlapping functions of Sufu and Kif7 in Hh

signaling during chondrocyte development. Recent studies established that Sufu and Kif7 are

evolutionarily conserved regulators of Gli transcription factors and that they both play negative

regulatory roles in Hh signaling during early mouse embryogenesis (Cheung et al., 2009; Cooper

et al., 2005; Svard et al., 2006). There is evidence suggesting that Sufu and Kif7 are also required

for high levels of Hh pathway activity (Chen et al., 2009), but the underlying mechanism is not

defined. Here I provide genetic and molecular data indicating that Sufu is a major negative

regulator of the Hh pathway in the growth plate and that Kif7 plays dual roles in controlling Ihh

signaling and chondrocyte development (Fig. 2-10A).

Phenotypic analysis of conditional knockout mice indicates that Sufu and Kif7 normally

play opposing roles in Ihh signaling in the developing chondrocytes. Sufu-deficient chondrocytes

showed augmented Hh pathway activity, increased proliferation and delayed differentiation. In

contrast, Kif7-deficient chondrocytes exhibited lower Hh pathway activity, a decrease in

proliferation, and an expansion of the hypertrophic zone. This could be due to the negative effect

of proliferation or the stimulatory effect on chondrocyte hypertrophy. I showed that Sufu protein

levels are elevated in Kif7-deficient chondrocytes and that reduction of Sufu gene dosage

restores Hh pathway activity and growth plate development. These results suggest that a major

role for Kif7 in the growth plate is to maintain Hh signaling activity by lowering the level of

Sufu, the major negative regulator of the pathway. Importantly, Kif7 also plays a negative role in

suppressing Hh signaling activity in Sufu-deficient chondrocytes as revealed by further pathway

augmentation in Sufu;Kif7-deficient chondrocytes. This partly explains why the Hh pathway is

40

not fully activated in Sufu-deficient chondrocytes and why the Sufu knockout growth plate

phenotype is not as severe as those of Ptch1 knockout mice (Mak et al., 2008). Interestingly, I

also found that Hh pathway activation in Sufu; Kif7-deficient chondrocytes is not as robust as

those observed in Ptch1-deficient chondrocytes. It remains to be determined whether this is due

to the requirement of Sufu and/or Kif7 for full transcriptional activity of Gli proteins, or related

to the ability of Ptch1 to sequester Hh ligands in the extracellular environment. Nevertheless, our

data clearly revealed a cross-regulation of Sufu and Kif7 in Ihh signaling during chondrocyte

development and demonstrated that Kif7 can positively modulate Hh pathway activity through

down-regulation of Sufu. These results also represent to date the first genetic evidence that Sufu

and Kif7 play overlapping regulatory roles in the negative control of the Hh signaling pathway.

Hh pathway activation promotes degradation of Sufu in some cancer cell lines (Yue et al.,

2009). Our results here show that Kif7 plays a key role in controlling the stability of Sufu protein

in chondrocytes. Strikingly, removal of one copy of Sufu could restore normal chondrocyte

development in Kif7-deficient growth plates indicating that the control of Sufu protein/activity

by Kif7 is a critical regulatory step in chondrocyte proliferation and differentiation. Recent

studies suggest that Hh stimulation promotes the dissociation of Sufu-Gli protein complexes at

the ciliary tip and that this dissociation is important for Gli activation (Humke et al., 2010;

Tukachinsky et al., 2010). Interestingly, I found that Sufu is mostly excluded from the ciliary tip

in wild-type chondrocytes, whereas Sufu appear to be stabilized in the ciliary tip in the absence

of Kif7. This is consistent with the observation that Hh pathway activity is reduced in Kif7-

deficient chondrocytes (due to inefficient dissociation of the Sufu-Gli complexes) and supports

the notion that Kif7 plays a direct or indirect role in the Hh-stimulated dissociation of Sufu-Gli

protein complexes and Gli activation. However, whether the ciliary tip localization contributes to

41

the stabilization of Sufu in Kif7-deficient chondrocytes or is related to the increased Sufu protein

level awaits further investigation. Nonetheless, our results clearly demonstrated that Sufu is a

regulatory target of Kif7 in Ihh signaling during chondrocyte development.

Previous in vitro data show that Hh stimulation promotes the translocation of Kif7 to

the tip of the cilia (Liem et al., 2009). Here I found that Kif7 localizes to the ciliary tip in both

the resting and proliferating chondrocytes in vivo (data not shown). Since the Hh pathway is

inactive (as indicated by the lack of Hh target gene expression) in the resting zone, these results

suggest that Kif7 might have a functional role in the primary cilium, even in the absence of Hh

ligand activation. Such a role is likely important in the growth plate, allowing for the normal

regulation of Gli proteins and other Hh pathway components, such as Sufu. This also hints that

dynamic localization of Kif7 in the primary cilium itself is likely not the key control in

regulating Hh ligand-mediated transcription in the growth plate chondrocytes. In embryonic

fibroblasts, Gli2 and Gli3 do not localize to the cilium in the absence of Sufu (Chen et al., 2009).

However, I found that in the Sufu-deficient chondrocytes, both Gli2 and Gli3 can still be found in

the cilia. This supports the concept that Sufu is not required for the Gli proteins to localize to the

cilia (Tukachinsky et al., 2010) and also raises the possibility that other proteins or processes in

the cilium are important for the regulation of Gli proteins, and their ability to regulate

transcription.

During endochondral bone development, the coordinated differentiation of growth plate

chondrocytes regulates the pace of long bone growth. Hh signaling plays a critical role in this

process, but how the cells are able to escape their proliferative state and undergo terminal

differentiation is unclear. The proliferation and differentiation defects observed in Kif7- and

Sufu-deficient mice suggest that Sufu and Kif7 expression plays an important role in how cells in

42

various regions of the developing growth plate process Hh signals as they progress from the

resting to hypertrophic zones. In wild-type mice, Sufu and Kif7 are highly expressed in the

periarticular/resting region of the growth plate where minimal Ptch1 expression was observed

However, in the proliferating and hypertrophic regions, the level of expression of Sufu and Kif7

decreases and Ptch1 expression increases. Thus, the gradient of expression of Sufu and Kif7

negatively correlates with Ptch1 expression and Hh activity. Deletion of both Sufu and Kif7

resulted in a significant expansion of the Ptch1 expression, notably in the resting region of the

growth plate, suggesting that the level of Sufu and Kif7 plays an important role in how cells

process Hh signals in the growth plate. Intriguingly, the expression pattern of Sufu and Kif7 is

very similar to the expression pattern of Parathyroid hormone-like protein (Pthlh; previously

known as PTHrP), which raises the possibility that interaction between those molecules may

control how Pthlh regulates Hh signaling activity during development.

Taken together, I show here a novel mechanism by which Sufu and Kif7 interact to

mediate Hh signaling in the growth plate (Fig. 2-10, B). In wild-type chondrocytes, Kif7

positively regulates Gli-mediated transcription by downregulating Sufu protein levels, but also

inhibits Gli-mediated transcription through a Sufu-independent mechanism (Fig. 2-10Aa). Its

role in the primary cilium allows for normal Sufu-Gli complex localization and processing (Fig.

2-10B). In Sufu-deficient chondrocytes, the regulatory role of Kif7 on Sufu is lost; Gli protein

localization in the cilium does not differ much from the situation in wild-type chondrocytes,

resulting in a sub-maximal level of Hh pathway activation (Fig. 2-10Ab). In Kif7-deficient

chondrocytes, Sufu acts unopposedly as a negative regulator of Gli-mediated transcription, and

there is an increased level of Sufu-Gli complexes in the primary cilium, resulting in a greater

inhibition of Hh signaling than in the wild-type situation (Fig. 2-10Ac). In the absence of both

43

Sufu and Kif7, Gli proteins will not be bound to Sufu and prevented from becoming

transcriptionally active. The inhibitory effect of Kif7 is relieved, leading to further augmentation

of the Hh pathway activity (Fig. 2-10Ad), showing that the regulation of Hh signaling by Kif7

and Sufu play critical roles in the growth plate.

44

2.5 Materials & Methods

Ethics Statement

A mouse protocol describing the experimental procedures used in the study was approved

by the Animal Care Committee of The Hospital for Sick Children.

Mice

The generation of Kif7-deficient mice was previously reported (Cheung et al., 2009).

Conditional Sufu-deficient mice (Col2a1-Cre;Sufuf/f

) were generated by crossing Col2a1-Cre

mice expressing Cre-recombinase under type II collagen regulatory elements specific to

chondrocytes with Sufu-floxed mice containing loxP sites flanking exons 4 to exon 8 of Sufu

(Pospisilik et al., 2010). Conditional Ptch1 deficient mice (Col2a1-Cre; Ptch1f/f

) were generated

from Ptch1-floxed mice, which contain loxP sites flanking exon 3 of Ptch1 (Adolphe et al.,

2006; Ellis et al., 2003). Embryonic mice were obtained from timed pregnancies and the

genotypes of the various mice were determined as described (Cheung et al., 2009; Ding et al.,

1999). In all cases, littermate mice were used as controls. The recombination efficiency in all

conditional mutants was confirmed through PCR, western analysis, and by examining the Cre-

drivers crossed with a Rosa-26 reporter line. All mice are on the 129/Sv background.

Skeletal staining

45

Mice were fixed in 95% ethanol after removal of skin and viscera. Bone samples were

incubated in Alcian Blue solution (15% Alcian Blue in 80% ethanol and 20% glacial acetic acid)

for 2-3 days at room temperature. Samples were then rehydrated and cleared in 1% KOH

overnight or till clear. Samples were stained with Alizarin Red solution (7.5% Alizarin Red in

1% KOH) for 1-2 days and immersed in glycerol for storage (Mau et al., 2007).

Microdissection of the growth plate

Hindlimbs were obtained from E18.5 mouse embryos. Sections of the growth plate were

dissected out using the assistance of a microscope followed by RNA isolation using Trizol

reagent (Invitrogen). RNA concentration was determined by Nanodrop. cDNA was synthesized

from 500 ng of total RNA using qScript cDNA SuperMix (Quanta Biosciences) for Real time

PCR analysis.

Histological analysis and Immunohistochemistry

Samples were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and

sectioned for histological evaluation. Sections were examined for Col10a1, a marker for

hypertrophic growth plate chondrocytes, by immunohistochemistry using previously reported

techniques and antibodies (Hu et al., 2006; Linsenmayer et al., 1988; Saika et al., 2004; Tiet et

al., 2006; Wang et al., 2000). The proximal tibial growth plate was used for all analysis to

minimize morphological variations due to anatomic location. Hematoxylin and eosin (H&E),

Safranin O and Alcian blue staining were performed using standard techniques. Proliferation was

46

evaluated by immunostaining using antibodies against Ki-67 (DakoCytomation M7249) at a 1:50

dilution and Phospho-H3 (Sigma) at a 1:200 dilution at 4 °C overnight. The proportion of

positively stained cells were calculated in the proliferative zone of the growth plate by counting

the number of positive and negative cells over 10X high powered field. Apoptotic cells were

detected by using an antibody against active Caspase-3 (Promega, Madison, WI, Cat # G7481)

and TUNEL assay as previously reported (Tiet et al., 2006). Positively stained cells were

analyzed in a similar manner as for Ki67 staining. Sufu protein was detected using a rabbit anti-

mouse antibody (Santa Cruz Biothechnology, sc-28847) incubated at a 1:100 dilution at 4 °C

overnight.

Western analysis and Co-immunoprecipitation

Western blot analysis was performed using standard protocols. Immunoblotting was

performed overnight at 4 °C with the following primary antibodies: Gli3 antibody from Santa

Cruz Biotechnology (1:800), actin antibody from Oncogene (1: 10000), Sufu antibody (Meng et

al., 2001; 1:3000), Kif7 antibody (Cheung et al., 2009; 1:1000), and Gli2 antibody (Hu et al.,

2006; 1:1000). Precipitation of Sufu protein was performed using Dynabeads Protein A

(Invitrogen following manufacture’s protocol.

Real-time quantitative PCR

RNA isolated from at least three independent experiments was analyzed by qRT-PCR in

triplicate for each treatment condition and primer set. The reactions were made up in TaqMan

47

Universal PCR master mix (Applied Biosystems) with TaqMan Gene Expression Assays for

mouse Gli1, Ptch1, Hhip1, Gli2, Gli3, Ihh, Kif7 and Sufu (Applied Biosystems). The gene

expression levels between samples were analyzed using the 2 ΔΔCt

method (Livak and

Schmittgen, 2001). Either GAPDH or β-actin (Applied Biosystems) was used as endogenous

control for target gene normalization.

Primary growth plate chondrocyte cultures

Chondrocyte isolation protocol was modified from previous published methods (Gosset

et al., 2008). Growth plates of the hindlimbs from E16.5 embryos were isolated and incubated in

collagenase type 4 (Worthington) solution (3mg/ml) for 45 min at 37°C incubator, under 5%

CO2 in a petri dish. Soft tissues were detached by pipetting. The growth plates were placed in a

clean petri dish with 0.5 mg/ml collagenase type 4 solution and were incubated overnight at

37°C. Collagenase type 4 solution containing chondrocytes was collected. Cells were washed

with PBS and seeded at a density of 8x103 cells per cm

2.

Measuring protein turnover

Cells were cultured till reach confluency before cycloheximide (20 µM) (Calbiochem)

treatment to block de novo protein synthesis for the time indicated. At the end of each time point,

cells were collected for western analysis.

48

Immunofluorescence and in situ hybridization

Samples were fixed in 4% paraformaldehyde overnight, embedded in paraffin and

sectioned. Sections (5 µm) of growth plate were cut parallel to the longitudinal axis of the bone.

The sections were stained with a monoclonal antibody against acetylated α-tubulin (clone 6-11b-

1, 1:1000; Sigma- -

tubulin (Dyomics647) (1:100; Abcam), and/or a monoclonal antibody against Sufu (1:100;

Abcam), and/or a polyclonal antibody against Kif7 (1:100) at 4°C overnight. Secondary

antibodies conjugated with Alexa Fluor 488 (1:100; Invitrogen) and TRITC (1:100 Jackson

ImmunoResearch Laboratories) were applied for 45 minutes to detect the primary antibody.

Finally the sections were mounted in 4 ,6-diamidino-2-phenylindole (DAPI) containing

Vectashield (Vector Laboratories). Confocal images were acquired using a spinning disc

confocal microscope. In situ hybridization of embryonic sections was performed as described

previously (Mo et al., 1997).

Statistical analysis

For all of the data, the mean, 95 % confidence interval, and standard deviation were

calculated for each condition, and Student’s t test was utilized to compare data sets. A p value of

< 0.05 was used as a threshold for statistical significance.

49

2.6 Figures

Figure 2-1 Sufu is differentially expressed in the growth plate.

Figure 2-1. Sufu is differentially expressed in the growth plate. (A, B) Expression of Sufu and

Ihh in wild-type mouse growth plates at E18.5 assessed by qRT-PCR. (AC/RC: articular

chondrocytes/resting chondrocytes; PC/PRC: proliferating chondrocytes/prehypertrophic

chondrocytes; HC: hypertrophic chondrocytes) Data are shown as means and 95% confidence

intervals (n=3). (C) Immunohistochemical analysis of E16.5 wild-type mouse growth plates

showed that Sufu is highly expressed by in AC/RC and PC/PRC regions and fewer than 10 % of

the HC are stained positive for Sufu. (D) In situ hybridization analysis of Sufu expression in

wild-type mouse growth plate at E16.5. (E) Analysis of immunohistochemical studies. ~25% of

50

the cells in the AC/RC and PC/PRC regions are Sufu-positive. Less than 10% of the HC are

Sufu-positive. Data are shown as means and 95% confidence intervals (n=3).

Figure 2-2 Generating chondrocyte-specific knockout of Sufu.

51

Figure 2-2. Generating chondrocyte-specific knockout of Sufu. (A) A lack of

immunohistochemical staining for Sufu was found in Col2a1-Cre; Sufuf/f

growth plate

demonstrating efficient deletion of Sufu in chondrocytes. (B) Effective deletion of Sufu in growth

plate chondrocytes was also verified using western analysis. (C) a and b: Immunohistochemical

staining of Sufu in the growth plates of wild-type and Col2a1-Cre; Sufuf/f

mice at E16.5; c and d:

magnified images of the indicated areas in a and b, respectively (same images used in A). Due to

the loss of glycogen and lipid during tissue preparation, the chondrocyte (Ch) display little more

than the nucleus residing in a lacuna (L). (D) P10 Col2a1-Cre; Sufuf/f

mutant mice showed

significant reduction in body length compared with wild-type littermates. (E and F) Analysis of

P10 Col2a1-Cre; Sufuf/f

mutant mice showed significant reduction in body length and body

weight compared with littermate wild-type controls. Data are shown as means with 95%

confidence intervals indicated. (n=3). *P<0.05.

52

Figure 2-3 Skeletal phenotype analysis of Col2a1-Cre; Sufuf/f.

53

Figure 2-3. Skeletal phenotype analysis of Col2a1-Cre; Sufuf/f

. (A) Alcian Blue/ Alizarin Red

staining showed a delay in ossification in the P10 tibial bones and P0 lumbar vertebrae of the

Col2a1-Cre; Sufuf/f

mice. (B) Safranin O staining revealed an expansion of the proliferative zone

(PZ), a decrease in the length of the hypertrophic zone (HZ), and a delayed secondary

ossification (SOC) formation in the tibia of P10 Col2a1-Cre; Sufuf/f

mice (upper panels). An

increased length of the PZ and a reduction of the HZ were also found in the P10 mutant vertebral

growth plates as revealed by H&E staining (lower panels). (C) H&E staining revealed an

expansion of the proliferative zone, which is represented by the length from the end of the tibia

to the beginning of the hypertrophic zone (black bar), and a reduction of the hypertrophic zone

(red bar) in the Sufu-deficient tibial growth plates compared with wild-type littermates at E16.5.

Lower panel: magnified images of the hypertrophic zone (red bar) seen in the upper panel. (D)

Analysis of skeletal staining of Col2a1-Cre; Sufuf/f

mice exhibited delayed ossification

demonstrated by the decreased percentage of the length of the mineralized zone to the length of

the tibia. Data are shown as means with 95% confidence intervals indicated. (n=3). *P<0.05. (E

and F) Deletion of Sufu in chondrocytes resulted in a significant increase in the percentage of

Ki67-positive cells (indicated by red arrows at lower panel) in the E16.5 growth plate. Data are

shown as means with 95% confidence intervals indicated. (n=3). *P<0.05. (G) No significant

difference was found in apoptosis rate in the E16.5 Sufu-deficient growth plates demonstrated by

TUNEL staining and active caspase-3 staining. Positive cells were indicated by red arrows. Data

are shown as means with 95% confidence intervals indicated. (n=3). *P<0.05.

54

Figure 2-4 Sufu negatively regulates growth plate chondrocyte differentiation.

55

Figure 2-4. Sufu negatively regulates growth plate chondrocyte differentiation. (A-C)

Histological and immunohistochemical analysis of the E16.5 mouse tibia showed the length of

the hypertrophic zone (red bar), which is characterized by Col10a1 staining, is reduced in

Col2a1-Cre; Sufuf/f

embryos compared to wild-type littermates. An increased size of the

proliferative zone, which represented by the length from the end of the tibia to the beginning of

the Col10a1 expression zone (black bar) was found in the Col2a1-Cre; Sufuf/f

embryos. This

phenotype resembles the phenotype displayed by deletion of Ptch1, another repressor of Hh

signaling, although less severe. Data are shown as means and 95% confidence intervals (n=3). *

p<0.05. (D) Expression of Hh target genes, Gli1, Ptch1, and Hhip1, were assessed by qRT-PCR.

Data are shown as means and 95% confidence intervals (n=3-6). * p<0.05. (E) Protein levels of

Gli2, full-length Gli3 (Gli3FL), and repressor form of Gli3 (Gli3R) in wild-type and Sufu-

deficient growth plates were assessed by western analysis. Actin was used as loading control.

Sufu-deficient chondrocytes exhibited elevated Gli2 level and an increase in the Gli3FL:Gli3R

ratio (*: non-specific band).

56

Figure 2-5 In situ hybridization analysis of Ptch1 and Col10a1 expression in various genotypes.

Figure 2-5. In situ hybridization analysis of Ptch1 and Col10a1 expression in various genotypes.

(A-D) In situ hybridization for Ptch1 expression in the growth plates of wild-type, Col2a1-Cre;

Sufuf/f

, Col2a1-Cre; Kif7f/f

, and Col2a1-Cre; Sufuf/f

; Kif7f/f

mice at E16.5; E-F: In situ

hybridization for Col10a1 expression in the growth plates of wild-type, Col2a1-Cre; Sufuf/f

,

Col2a1-Cre; Kif7f/f

, and Col2a1-Cre; Sufuf/f

; Kif7f/f

mice at E16.5. Studies using a sense probe as

control showed no hybridization.

57

Figure 2-6 Kif7 inactivation resulted in opposite effects on chondrocyte proliferation and differentiation to those of Sufu

inactivation.

58

Figure 2-6. Kif7 inactivation resulted in opposite effects on chondrocyte proliferation and

differentiation to those of Sufu inactivation. (A) Immunohistochemical analysis of the tibia of

E16.5 wild-type embryos showed that Kif7 is highly expressed in the AC/RC region, and its

expression progressively decreases as chondrocytes differentiate. (B) Expression of Kif7 in wild-

type mouse growth plates at E16.5 assessed by qRT-PCR. Data are shown as means with 95%

confidence intervals as indicated (n=3). (C) In situ hybridization analysis of Kif7 expression in

the growth plate. (D) Safranin O and Col10a1 staining revealed an expansion of the hypertrophic

zone (red bar), and reduced length of the proliferative zone (black bar) in the Kif7 knockout mice

(Kif7-/-

) compared to wild-type littermates. (E) Alcian Blue/ Alizarin Red staining of wild-type

and Col2a1-Cre; Kif7f/f

littermates at E18.5 and P10. Col2a1-Cre; Kif7f/f

mice exhibited no gross

abnormalities in skeletal development. (F, G) An increase in the length of the hypertrophic zone

and a reduction in the length of the proliferative zone were found in Kif7-/-

and Col2a1-Cre;

Kif7f/f

mice compared to wild-type littermates. Data are shown as means and 95% confidence

intervals (n=3). * p<0.05. (H) H&E staining on the tibial sections of P10 wild-type and Col2a1-

Cre; Kif7f/f

mice (SOC: secondary ossification center; PZ: proliferative zone; HZ; hypertrophic

zone). (I) A significant decrease in the percentage of proliferating cells was found in Kif7-/-

and

Col2a1-Cre; Kif7f/f

growth plates as measured by immunohistochemical staining for Ki67. Data

are shown as means and 95% confidence intervals (n=3). * p<0.05. (J) Sufu and kIf7 possess

positive and negative effects on growth plate chondrocyte proliferation, respectively,

demonstrated by immunohistochemical staining for Phospho H3.

59

Figure 2-7 Increased level of Sufu in Kif7-deificent chondrocytes.

Figure 2-7. Increased level of Sufu in Kif7-deficient chondrocytes. (A) Sufu protein levels in

wild-type and Kif7-deficient growth plates were assessed by western analysis. Actin was used as

loading control. (B) Sufu mRNA expression level was found comparable with the wild-type

controls. Data are shown as means and 95% confidence intervals (n=3). (C) A similar Kif7

protein level was found in Sufu-deficient chondrocytes. (D) The mRNA expression level of Kif7

remained comparable with the wild-type controls. (E, F) Western analysis of Sufu in wild-type

and Kif7-deficient chondrocytes following 20µM cycloheximide (CHX) treatment. (G, H)

Western analysis of Sufu in wild-type and Kif7-deficient chondrocytes following 25µM MG132

treatment. Actin was used as loading control.

60

Figure 2-8 Increased level of Sufu-Gli complexes in Kif7-deificent chondrocytes.

61

Figure 2-8. Increased level of Sufu-Gli complexes in Kif7-deficient chondrocytes. (A)

Fluorescence micrographs of cilia from wild-type, Col2a1-Cre; Sufuf/f

, and Col2a1-Cre; Kif7f/f

growth plate chondrocytes. Gli2 (ciliary tip localization of Gli2 is indicated by an asterisk), Gli3

(ciliary tip localization of Gli3 is indicated by double asterisks), Sufu (ciliary base localization of

Sufu is indicated by a yellow arrow; ciliary tip localization of Sufu is indicated by a green

arrow), and Kif7 (ciliary tip localization of Kif7 is indicated by a white arrow) are detected in the

green channel. Cilia are detected by staining against acetylated α-tubulin (red channel).

Centrioles are detected by staining against γ-tubulin (blue channel) to label the base of the cilia.

(B-D) 25-50 ciliated cells from the proliferative zone of wild-type, Sufu-deficient and Kif7-

deficient growth plates were examined for Gli2, Gli3, Sufu, and Kif7 ciliary localization. Data

are shown as percentages. (E, F) Western analysis of total Gli2 protein and Gli2 protein

immunoprecipitated (IP) with anti Sufu antibody from control (C), cyclopamine- (Cy),

purmorphamine- (Pur), and Shh-treated primary chondrocyte cultures (*: rabbit IgG band). The

level of total Gli2 is comparable in all samples. The level of Gli2 co-immunoprecipitated with

Sufu is similar in cyclopamine-treated and control cells. Significantly less Gli2 co-

immunoprecipitates with Sufu in purmorphamine- and Shh-treated chondrocyte cultures. Data

are shown as means and 95% confidence intervals. ** p<0.01.

62

Figure 2-9 A dual function for Kif7 in Hh signaling modulation.

63

Figure 2-9. A dual function for Kif7 in Hh signaling modulation. (A-C) Histological and

immunohistochemical analysis of the mouse tibial growth plates. Upper panel: Alcian blue

staining on E16.5 tibial sections. Lower panel: Col10a1 immunostaining on E18.5 tibial sections

(black bar: proliferative zone; red bar: hypertrophic zone). Data are shown as means and 95%

confidence intervals (n=3). * p<0.05. (D) Expression of Gli1, Ptch1, and Hip1 in mouse growth

plates of various mutants at E16.5 assessed by qRT-PCR. Data are shown as means and 95%

confidence intervals (n=3-6). * p<0.05. See also Figure S1-3.

64

Figure 2-10 Proposed model for how Sufu and Kif7 regulate Hh signaling in growth plate chondrocytes.

65

Figure 2-10. Proposed model for how Sufu and Kif7 regulate Hh signaling in growth plate

chondrocytes. (A) Schematic representation of Hh signaling regulation in chondrocytes. a) In

wild-type cell, Sufu acts as a repressor in controlling Hh pathway activity. Kif7 possesses dual

functions in regulating Gli transcription activity. b) In the absence of Sufu, Kif7 acts as a

repressor, resulting in a submaximal level of Hh pathway activation. c) Loss of Kif7 resulted in

increased Sufu activity, resulting in reduced Hh pathway activity. d) Further augmentation of the

Hh pathway activity is found in the absence of both Sufu and Kif7. (B) Schematic representation

of Kif7-mediated dissociation of Sufu-Gli protein complexes at the primary cilium. Kif7 plays a

functional role at the tip of the primary cilium in growth plate chondrocytes; it positively

regulates Hh signaling activity via promoting the dissociation of Sufu-Gli complexes, leading to

Gli-mediated transcriptional activation. Kif7 also function negatively in Hh signaling possibly

through binding with Gli proteins in the cytoplasm.

66

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Chapter 3 Sufu mediates the effect of Pthlh on chondrocyte

differentiation in the growth plate

Shu-Hsuan C. Hsu, Xiaoyun Zhang, Chi-Chung Hui, and Benjamin A. Alman

All experiments and analysis described in this chapter were conducted by Shu-Hsuan C. Hsu

with the exception of the western analysis by Xiaoyun Zhang. This study is submitted to EMBO

Reports.

71

3.1 Summary

Growth plate chondrocytes undergo a coordinated process of proliferation and

differentiation regulating normal long bone growth. Parathyroid hormone-like hormone (Pthlh)

acts to regulate chondrocyte hypertrophic differentiation and the activation of hedgehog (Hh)

signaling regulated Gli transcription factors. The mechanism by which Pthlh regulates Hh

signaling is incompletely elucidated. Pthlh downregulates Gli transcriptional activity, and

inhibits hypertrophic differentiation in the growth plate chondrocytes. In the absence of

Suppressor of fused (Sufu), Pthlh treatment resulted in upregulated Hh activity and increased

size of the hypertrophic zone. Pthlh treatment resulted in phosphorylation of Sufu and increased

Sufu stability in wild-type chondrocytes through a protein kinase A (PKA)-mediated mechanism.

In contrast, the regulation of Pthlh on Gli transcriptional activity and chondrocyte differentiation

was independent of Kinesin family member 7 (Kif7), another mediator in the Hh signaling

cascade. Thus, Sufu is a critical regulator in the ability of Pthlh to regulate Gli transcription

activity and chondrocyte hypertrophic differentiation. Pthlh regulates Hh signaling activity and

chondrocyte hypertrophy by promoting Sufu stability through PKA-mediated phosphorylation,

allowing Gli2 degradation and Gli3 processing to a repressor form (Gli3R) in the growth plate

chondrocytes.

72

3.2 Introduction

Long bones grow through a process of endochondral ossification, in which growth plate

cartilage undergoes a coordinated process of proliferation and differentiation, ultimately

resulting in replacement by osteoblasts and new bone. Two signaling pathways that play a

critical role coordinating growth plate chondrocyte differentiation are Pthlh (also called PTHrP)

and Indian Hedgehog (Ihh). They act in a feedback loop, regulating each other’s activity. Pthlh

inhibits chondrocyte hypertrophic differentiation, while Ihh regulates the onset of hypertrophic

differentiation, in part by signaling the periarticular chondrocytes to upregulate the expression of

Pthlh. Ihh also regulates chondrocyte proliferation and induces ossification of the perichondrium

in a Pthlh-independent manner. These two pathways interact with each other at several levels in

their signaling cascades (Karp et al., 2000; Kobayashi et al., 2002; Lanske et al., 1996; Minina et

al., 2001).

Hh signaling is activated in response to an appropriate Hh ligand binding to the Patched 1

(Ptch1) receptor. This binding leads to Smoothened (Smo) protein activation, ultimately resulting

in the transcription of target genes. In Drosophila, Hh signaling activates the transcription factor

Cubitus interruptus (Ci). In the absence of Hh binding, Ci is cleaved to a transcriptional

repressor, while upon Hh binding, Smo activation allows uncleaved Ci to translocate into the

nucleus where it functions as a transcriptional activator. In mammals, there are three

transcription factors that mediate Hh signaling, Gli1, Gli2 and Gli3 (Jiang & Hui, 2008). Data

from studies in mice in the context of the development of major organ systems including the

limb suggest that Gli1 is functionally redundant. Gli2 is not processed and as such functions as

73

an activator, while Gli3 is processed similar to Ci, and can act as an activator or repressor of Hh-

mediated transcriptional activation (Bai et al., 2004; Ding et al., 1998; Mo et al., 1997;

Motoyama et al., 1998; Park et al., 2000). I elucidated the role of Sufu and Kif7, essential

regulators of Hh signaling, in the growth plate (Hsu et al., 2011; see also chapter 2). Sufu acts as

a repressor of the Hh signaling pathway (Cooper et al., 2005; Svard et al., 2006), while Kif7

functions both positively and negatively in the Hh signaling (Chen et al., 2009; Cheung et al.,

2009; Hui and Joyner, 1993). Kif7 positively regulates Hh signaling activity in part by regulating

Sufu protein level and the exclusion of Sufu-Gli complexes from the primary cilium (Hsu et al.,

2011). Thus, Sufu and Kif7 play important roles regulating Hh signaling activity and

chondrocyte differentiation.

Previous studies show that Pthlh and the transcription factors Gli2 and Gli3 interact

during growth plate chondrocyte differentiation and proliferation (Mau et al., 2007). Limb

explant cultures showed that Pthlh treatment inhibited Col10a1 expression, a marker of growth

plate chondrocyte differentiation, and increased chondrocyte proliferation. This effect was

substantially enhanced in Gli2-/-

limbs, was absent in Gli3-/-

limbs, and was only partially

inhibited by Hh ligand blockade. Pthlh negatively regulates Gli-mediated transcription in cell

cultures, and controls the level of the repressor form of Gli3 (Gli3R) in a PKA-dependent

manner (Mau et al., 2007; Miao et al., 2004; Tukachinsky et al., 2010; Tuson et al., 2011;

Vortkamp et al., 1996; Zeng et al., 2010). Thus, the interaction between Pthlh and Gli

transcription factors plays an important role regulating growth plate chondrocyte differentiation.

Sufu and Kif7 are critical mediators of Hh signaling and the regulation of chondrocyte

hypertrophic differentiation; they are highly expressed by articular and resting chondrocytes in

the growth plate, the same region as where Pthlh is expressed (Hsu et al., 2011). This co-

74

expression pattern raises the possibility that Pthlh and these Hh signaling intermediates interact

in these growth plate cells. Furthermore, since Sufu and Kif7 mediate Hh signaling and regulate

chondrocyte hypertrophic differentiation, I examined how the interaction of Pthlh, Sufu, and

Kif7 in the growth plate regulates chondrocyte hypertrophic differentiation.

75

3.3 Results

3.3.1 Sufu regulates the effect of Pthlh on chondrocyte differentiation

To determine if Sufu mediates the effect of Pthlh on growth plate chondrocyte

differentiation, I examined the effects of Pthlh treatment on chondrocyte hypertrophic

differentiation in E16.5 tibial explants from wild-type and Sufu-deficient mice (Col2a1-Cre;

Sufuf/f

). I found that Pthlh treatment reduced the length of the hypertrophic zone (represented by

Col10a1 staining) in explants from wild-type limbs (Fig. 3-1A, B). This finding is consistent

with the known inhibitory role of Pthlh on chondrocyte hypertrophic differentiation.

Unexpectedly, Pthlh treatment of Sufu-deficient explants resulted in an expansion of the

hypertrophic zone (Fig. 3-1A, B). Thus, Sufu is required for the normal inhibitory effect of Pthlh

on growth plate chondrocyte differentiation. Pthlh also possess a stimulatory effect on

chondrocyte proliferation (Beier et al., 2001). I found that Pthlh treatment resulted in an

expansion of the proliferative zone (represented by the length from the end of the tibia to the

beginning of the hypertrophic zone) in wild-type limbs (Fig. 3-1A, C). Similar increase in the

proliferative zone was observed in the Pthlh-treated Sufu-deficient limbs (Fig. 3-1A, C); showing

that in contrast to its role in regulating chondrocyte hypertrophic differentiation, Sufu is not

required for Pthlh to activate growth plate chondrocyte proliferation.

The effects of Pthlh on normal growth plate chondrocytes is primarily mediated by PKA

(Guo et al., 2002; Mau et al., 2007; Wang et al., 2000). A PKA-specific inhibitor H-89 abolished

the effects of Pthlh on both wild-type and Col2a1-Cre; Sufuf/f

explants (Fig. 3-1A-C). Taken

76

together, the data suggest that Pthlh signaling inhibits chondrocyte hypertrophic differentiation

through the activation of PKA, and it functions upstream of Sufu.

3.3.2 Kif7 is not required for Pthlh to inhibit chondrocyte differentiation

Kif7 plays a role in controlling Hh activity through restricting Sufu function in the

growth plate chondrocytes (Hsu et al., 2011). To determine if Kif7 is required for the effect of

Pthlh on chondrocyte differentiation, I performed tibial explants from E16.5 Col2a1-Cre; Kif7f/f

embryos. Similar to wild-type explants, Pthlh exerts an inhibitory effect on chondrocyte

hypertrophic differentiation in Kif7-deficient limbs (Fig. 3-2A, B), showing that Pthlh negatively

regulates chondrocyte hypertrophic differentiation through a Kif7-independent mechanism. In

growth plate chondrocytes, Kif7 demonstrates a negative function in Hh signaling in the absence

of Sufu (Hsu et al., 2011). Since Kif7 plays a different role regulating Hh pathway activity in the

absence of Sufu, I examined mice lacking both Sufu and Kif7. Pthlh treatment resulted in an

expansion of the hypertrophic zone in these mice lacking both Sufu and Kif7 (Fig. 3-2A, B),

similar to those observed in Col2a1-Cre; Sufuf/f

explants (Fig. 3-1A, B), indicating that Sufu is

indeed required for the inhibitory effect of Pthlh on chondrocyte hypertrophic differentiation.

3.3.3 Pthlh regulates Sufu protein level

Given the important role of Sufu in Pthlh-mediated regulation of chondrocyte

hypertrophy, I set out to investigate how Pthlh and Sufu might interact. To investigate whether

77

Sufu is controlled in response to Pthlh signaling activation, I analyzed Sufu levels in Pthlh-

treated wild-type explant cultures. In wild-type chondrocytes, Pthlh treatment resulted in a

significant increased level of Sufu protein. The increase of Sufu in Pthlh-treated cells is likely

due to post-translational regulation because the level of Sufu transcript in these cells remained

comparable with that of the controls as determined by qRT-PCR analysis (Fig. 3-3A, B). As

expected, Sufu protein was not found in cell lysates derived from Col2a1-Cre; Sufuf/f

explants,

demonstrating efficient deletion of Sufu in mutant chondrocytes (Fig. 3-3A).

Phosphorylation of Sufu protein by PKA can positively regulate its stability in cultured

fibroblasts (Chen et al., 2011). Since Pthlh functions mainly through PKA in the growth plate

chondrocytes, it is possible that Pthlh promotes phosphorylation of Sufu, leading to its

stabilization. To determine whether Pthlh stimulates Sufu phosphorylation in chondrocytes,

primary chondrocyte cultures were treated with Pthlh, and the level of serine phosphorylation of

Sufu was quantified using western analysis of phosphor-serine followed by immunoprecipitation

with Sufu-specific antibodies. There was a higher level of serine phosphorylation of Sufu in the

Pthlh-treated chondrocytes compared to the untreated controls (data not shown). Taken together,

these data suggest that, similar to those observed in cultured fibroblasts, Pthlh activation

promotes phosphorylation of Sufu and stabilizes Sufu protein in the chondrocytes.

3.3.4 Sufu is required for the ability of Pthlh to process Gli transcription factors

One mechanism by which Pthlh negatively regulates Gli-mediated transcription is

through processing of Gli3 (Mau et al., 2007). To investigate the interaction between Pthlh and

Gli transcription factors, I examined the levels of Gli2, full-length Gli3 (Gli3FL), and truncated

78

repressor form of Gli3 (Gli3R) in wild-type and Col2a1-Cre; Sufuf/f

growth plate chondrocytes

with or without the treatment of Pthlh. qRT-PCR analysis revealed comparable levels of Gli2

and Gli3 mRNA in wild-type and Sufu-deficient chondrocytes (Fig. 3-4A). In wild-type limbs,

Pthlh treatment resulted in a decreased level of Gli2. However, this effect was not seen in the

Sufu-deficient chondrocytes (Fig. 3-4B). Consistent with previous data, Western analysis

revealed a decrease in the ratio of full-length versus repressor form of Gli3 (Gli3FL:Gli3R) upon

Pthlh signaling activation in wild-type chondrocytes (Fig. 3-4C, D). However, Pthlh treatment

resulted in an increase in the ratio of Gli3FL:Gli3R in chondrocytes lacking Sufu (Fig. 3-4C, D).

To determine whether Sufu influences Pthlh-mediated regulation of Hh signaling activity,

primary chondrocytes from wild-type and Sufu-deficient mice were treated with Pthlh and the

expression of Gli1 and Ptch1 was analyzed using qRT-PCR. Consistent with the repressor role

of Sufu in Hh signaling pathway, significant increases in Gli1 and Ptch1 expression levels were

found in Sufu-deficient chondrocytes. In wild-type cells, Pthlh treatment resulted in reduced

expression level in Gli1 and Ptch1. In the absence of Sufu, Pthlh treatment poses a positive

effect on Gli1 and Ptch1 expression (Fig. 3-4E, F), which suggests that Sufu is required for the

inhibitory function of Pthlh on Hh signaling activity in the chondrocytes.

79

3.4 Discussion

Here, I found that Sufu is specifically required for the inhibitory effect of Pthlh on

chondrocyte hypertrophic differentiation. Because its absence results in a reversal of the sequels

of Pthlh stimulation of chondrocyte hypertrophic differentiation, it serves as a molecular switch

in Pthlh function. In contrast to the role I found for Sufu, Kif7 is not required for Pthlh to

influence chondrocyte hypertrophy in the growth plate. Thus, Sufu appears to be a critical link

between Hh and Pthlh signaling in the control of chondrocyte hypertrophic differentiation, and a

number of molecules play important roles regulating Sufu stability, which in turn is likely a

central node in Hh signaling activation.

Phosphorylation of Sufu through PKA activation promotes Sufu protein stability (Chen et

al., 2011). Here, I found that Pthlh increases Sufu protein level as well as its phosphorylation

level. Thus, our data support the notion that phosphorylation stabilizes Sufu and Pthlh plays a

role regulating this phosphorylation in the growth plate. Intriguingly, Kif7 functions positively

in regulating Hh pathway activity through restricting Sufu level (Hsu et al., 2011). Therefore,

the regulation of Sufu level may be a common mechanism regulating Hh-mediated transcription.

In the absence of Hh ligand, Sufu sequesters Gli proteins in the cytoplasm, preventing Hh

pathway activation (Barnfield et al., 2005; Ding et al., 1999; Kogerman et al., 1999; Murone et

al., 2000). Sufu also functions to promote full length Gli3 to be processed into it repressor form

(Gli3R) in concert with GSK3ß (Kise et al., 2009). Here, I found that the stimulatory effect of

Pthlh on the formation of the Gli3R is absent in Sufu-deficient chondrocytes, which supports the

notion that Sufu plays an important role in Gli3 protein processing. Although, Pthlh inhibits Gli

80

transcriptional activity and chondrocyte hypertrophy independent of Gli2 (Mau et al., 2007), I

found that Pthlh promotes Gli2 degradation in wild-type chondrocytes. However, this effect of

Pthlh on Gli2 is not seen in chondrocytes lacking Sufu, suggesting that Sufu is required for this

process. Thus, these data suggest that Pthlh stimulates Gli2 degradation and the formation of

Gli3R through a Sufu-dependent mechanism. Taken together, I propose a model by which Sufu

mediates the effect of Pthlh signaling in the growth plate (Fig. 3-5). Pthlh positively regulates

Sufu protein stability in part through phosphorylation and promotes Sufu-mediated Gli2

degradation and Gli3 protein processing, which inhibits chondrocyte hypertrophic

differentiation.

Ihh and Pthlh maintain chondrocytes in a proliferative state and premature differentiation

occurs in the absence of a functional Ihh/Pthlh feedback loop (Karp et al., 2000; Kobayashi et al.,

2002; Lanske et al., 1996; Minina et al., 2001; Kronenberg, 2003; Long et al., 2001). However,

what controls the transition from a proliferative state to terminal differentiation is not elucidated.

Here, I found that Pthlh treatment delays hypertrophic differentiation in Kif7-deficient limb

explants, but not in mutant limb explants lacking Sufu (Col2a-Cre;Sufuf/f

as well as Col2a-

Cre;Sufuf/f

;Kif7f/f

). The requirement of Sufu for the effect of Pthlh on chondrocyte hypertrophic

differentiation raises the possibility that Sufu serves as the molecular switch regulating the

transition from proliferative to hypertrophic chondrocytes.

In contrast to the PKA-mediated action of Pthlh on chondrocyte hypertrophic differentiation,

its stimulatory effect on chondrocyte proliferation is not mimicking by PKA activation. Notably,

in the absence of Sufu, Pthlh treatment promotes chondrocyte proliferation and subsequently

increases the number of growth plate chondrocytes. Thus, the stimulatory effect of Pthlh on

chondrocyte proliferation is not dependent on Sufu, suggesting that Sufu is involved in the PKA-

81

mediated action of Pthlh. Taken together, Sufu appears to link Ihh and Pthlh signaling in the

control of chondrocyte proliferation versus differentiation. Indeed, I showed here that Sufu

protein expression is regulated by Pthlh. It is important to note that Sufu is highly expressed in

the articular and resting chondrocytes (Hsu et al., 2011). It is plausible that phosphorylation of

Sufu by Pthlh near the articular region acts to maintain the level of Sufu protein, thus, preventing

Hh pathway activation mediated by Ihh that is expressed by the prehypertrophic chondrocytes.

Therefore, the low level of Sufu in prehypertrophic and hypertrophic chondrocytes may be

involved in determining the transition from proliferation to differentiation.

Furthermore, a germline mutation of Sufu has been identified in a family of Gorlin

syndrome, which is characterized by a range of developmental abnormalities and increased risk

of developing of basal cell carcinoma (Pastorino et al., 2009). The pathogenesis of basal cell

carcinoma, which is the most common skin tumor, involves aberrant activation of the Hh

signaling pathway (Epstein, 2008). Elevated Hh signaling in basal cell carcinomas can be

attributed to loss of Ptch1, independent overexpression of Gli1 and Gli2 (Dahmane et al., 1997;

Gailani et al., 1996; Grachtchouk et al., 2000; Reifenberger et al., 2005). In mice, deletion of

one allele of Sufu resulted in a basal cell carcinoma-like neoplastic growth. Interestingly, Pthlh

expression does not seem to be altered in basal cell carcinomas (Philbrick et al., 1996). Thus, it is

tempting to speculate that Sufu functions as a tumor suppressor through mediating Gli

processing downstream of Pthlh signaling activation in the skin. Therefore, proper regulation of

Sufu-mediated Gli processing plays important role in both embryogenesis and tumorgenesis.

82

3.5 Materials & Methods

Ethics Statement



Mice

The generation of Kif7-deficient mice was previously reported (Cheung et al., 2009).

Conditional Sufu-deficient mice (Col2a1-Cre; Sufuf/f

) were generated by crossing Col2a1-Cre

mice expressing Cre-recombinase under type II collagen regulatory elements specific to

chondrocytes with Sufu-floxed mice containing loxP sites flanking exons 4 to exon 8 of Sufu.

Conditional Ptch1-deficient mice (Col2a1-Cre; Ptch1f/f

) were generated from Ptch1-floxed mice,

which contain loxP sites flanking exon 3 of Ptch1 (Adolphe et al., 2006; Ellis et al., 2003).

Embryonic mice were obtained from timed pregnancies and the genotypes of the various mice

were determined as described (Cheung et al., 2009; Ding et al., 1999). In all cases, littermate

mice were used as controls. The recombination efficiency in all conditional mutants was

confirmed through PCR, western analysis, and by examining the Cre-drivers crossed with a

Rosa-26 reporter line. All mice are on the 129/Sv background. A mouse protocol describing the

above experimental procedures was approved by the Animal Care Committee of The Hospital

for Sick Children.

83

Skeletal staining






Explant cultures

Hindlimb organ cultures from E16.5 embryos were established as previously described

(Ho et al., 2009; Lanske et al., 1996; Mau et al., 2007; Minina et al., 2001). i bs ere placed

on a ucleopore filter inside a ell of - ell tissue culture plate, ith the li b at the li uid air

interface. The li b e plants ere then cultured for days at in a 2 incubator in

DMEM containing 0.1% BSA, Vitamin C (50 mg/ ml), and Antibiotics-Antimycotic Solution

(Wisent). One limb from each embryo was treated with various agents whereas the other acted as

a control. 10-7

M Pthlh (Bachem, King of Prussia, Pennysylvania, USA) was added to activate

Pthlh signaling. The PKA inhibitor, H-89 dihydrochloride (Alexia Biochemicals), was used at

20µM. These reagents were used alone or in combination for two consecutive days, with media

changed each day replenishing the specific agent utilized. Each explant experiment was

undertaken three times or more using limbs from three different fetal mice and littermate

controls.

84







minimize morphological variations due to anatomic location. Safranin O staining was performed

using standard techniques. Proliferation was evaluated by immunostaining using antibodies

against Ki-67 and Phospho-H3 (DakoCytochemical M7249 (from Clone Tec-3) at a 1:50 dilution

or (from Sigma) at a 1:200 dilution) at 4 °C overnight. The proportion of positively stained cells

were calculated in the proliferative zone of the growth plate by counting the number of positive

and negative cells over 10X high powered field. Apoptotic cells were detected by using an

antibody against active Caspase-3 (Promega, Madison, WI, Cat # G7481) and TUNEL assay as

previously reported (Tiet et al., 2006). Positively stained cells were analyzed in a similar manner

as for Ki67 staining. Sufu protein was detected using a rabbit anti-mouse antibody (Santa Cruz

Biothechnology, sc-28847) incubated at a 1:100 dilution at 4 °C overnight.

Western analysis

Western blot analysis was performed using standard protocols. Immunoblotting was

performed overnight at 4 °C with the following primary antibodies: Gli3 antibody from Santa

Cruz Biotechnology (1:800), actin antibody from Oncogene (1: 10000), Sufu antibody (Meng et

85

al., 2001), Kif7 antibody (Cheung et al., 2009), and Gli2 antibody (Hu et al., 2006), and

phosphor-serine antibody from Cell Signaling (1:1000).

Real time quantitative PCR




mouse Gli1, Ptch1, Gli2, Gli3, and Sufu (Applied Biosystems). The gene expression levels

between samples were analyzed using the 2 ΔΔ t

method (Livak and Schmittgen, 2001). Either

GAPDH or β-actin (Applied Biosystems) was used as endogenous control for target gene

normalization.


Chondrocyte isolation protocol was modified from previously published methods (Gosset




clean petri dish with 0.5 mg/ml collagenase D solution and were incubated overnight at 37°C.

Collagenase D solution containing chondrocytes was collected. Cells were washed with PBS and

seeded at a density of 8*103 cells per cm

2.

86



calculated for each condition, and the Student’s t test was utilized to compare data sets. The

threshold for statistical significance was p < 0.05.

87

3.6 Figures

Figure 3-1 Sufu is required for the negative effect of Pthlh on chondrocyte differentiation.

88

Figure 3-1. Sufu is required for the negative effect of Pthlh on chondrocyte differentiation. (A)

Histological analysis of tibial explants from wild-type and Col2a1-Cre; Sufuf/f

mice in the

absence or presence of Pthlh or Pthlh+H89. (B) Relative percentage difference of the length of

the hypertrophic zone of Pthlh or Pthlh+H89 treated tibial explants compared to the controls. (C)

Relative percentage difference of the proliferative zone of Pthlh or Pthlh+H89 treated tibial

explants compared to the controls. Data are shown as means and 95% confidence intervals (n=3).

89

Figure 3-2 Kif7 is not required for the negative effect of Pthlh on chondrocyte differentiation.

Figure 3-2. Kif7 is not required for the negative effect of Pthlh on chondrocyte differentiation.

(A) Immunohistochemical staining for Col10a1 and Safranin O staining on tibial explants of

Col2a1-Cre; Kif7f/f

and Col2a1-Cre; Sufuf/f

; Kif7f/f

mice with or without Pthlh treatments. (B)

Relative percentage difference of the length of the hypertrophic zone of Pthlh-treated tibial

explants compared to the controls. Data are shown as means and 95% confidence intervals (n=3).

90

Figure 3-3 Pthlh increases Sufu protein level.

Figure 3-3. Pthlh increases Sufu protein level. (A) Pthlh treatment resulted in elevated level of

Sufu protein in wild-type growth plate chondrocytes. Effective deletion of Sufu in chondrocytes

was verified using western analysis. Actin was used as loading control. (Un: untreated) (B) qRT-

PCR analysis of Sufu expression levels in wild-type chondrocytes with or without Pthlh

treatment. (Un: untreated). Data is shown as means and 95% confidence intervals. * p<0.05.

91

Figure 3-4 Pthlh signaling activation promotes Gli2 and Gli3 protein processing via a Sufu-mediated manner.

92

Figure 3-4. Pthlh signaling activation promotes Gli2 and Gli3 protein processing via a Sufu-

mediated manner. (A) qRT-PCR analysis of the expression level of Gli2 and Gli3 in Wild-type

and Sufu-deficient chondrocytes. (B) Western analysis for Gli2 of wild-type and Sufu-deficient

chondrocytes with or without Pthlh. Actin was used as loading control. (C) Western analysis for

full-length Gli3 (Gli3FL) and truncated Gli3 (Gli3R) of wild-type and Sufu-deficient

chondrocytes with or without Pthlh treatments. (D) Graphical evaluation of the Gli3FL:Gli3R

ratio in wild-type and Sufu-deficient chondrocytes with or without Pthlh treatment. (E) qRT-PCR

analysis of Gli1 expression in wild-type and Sufu-deficient growth plate chondrocytes with or

without Pthlh treatment. (F) qRT-PCR analysis of Ptch1 expression in wild-type and Sufu-

deficient growth plate chondrocytes with or without Pthlh treatment. Data is shown as means and

95% confidence intervals. * p<0.05.

93

Figure 3-5 Proposed model for how Pthlh regulates Gli transcriptional factors through a Sufu-mediated mechanism.

Figure 3-5. Proposed model for how Pthlh regulates Gli transcriptional factors through a Sufu-

mediated mechanism. Pthlh promotes Sufu protein level through phosphorylation, and facilitates

Gli2 degradation and Gli3R formation, inhibiting chondrocyte hypertrophic differentiation in the

growth plate.

94

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423-34.

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Chapter 4 Conclusions & future directions

98

4.1 Summary

Although the signaling cascades in the regulation of the growth plate chondrocyte

development have been identified, we know little about the precise mechanism by which they

act. This study provides insight into a number of open questions in the Hh signaling field as well

as into the development of growth plate; how is the dual function of Kif7 mediated, how does

Kif7 control Sufu activity, how is Hh pathway activity regulated by Sufu and Kif7 in the context

of primary cilium, and how is Sufu activity coupled to Pthlh signaling? In order to develop

effective therapeutic approaches to target diseases caused by Hh pathway dysregulation,

revealing the molecular basis underlying the Hh signaling cascade in various tissues will be

necessary.

99

4.2 Sufu expression is tightly regulated by multiple factors in the growth

plate

4.2.1 Lessons from genetically modified mice and Pthlh activation

In chapter 2, I confirmed that Sufu serves as a negative regulator of the Hh pathway, and

it is differentially expressed in the growth plate. Kif7 promotes Hh signaling in chondrocytes by

restricting the inhibitory function of Sufu in part through a proteasome-mediated pathway. Kif7

also negatively regulates Hh activity through a Sufu-independent mechanism. Previous studies in

cultured fibroblasts have demonstrated that Hh signaling activation accelerates Sufu protein

turnover through promoting its degradation in the proteasome (Yue et al., 2009). These data

suggest that controlling Sufu protein stability serves as a common mechanism that connects the

action of Smo to downstream Gli-mediated transcription (Fig. 4-1).

In chapter 3, I demonstrated that Pthlh increases Sufu protein levels possibly through

phosphorylation (Fig. 3-3). Studies have shown that Sufu is phosphorylated at Ser-342 and Ser-

346 by GSK3ß and PKA, respectively, which leads to Sufu stabilization against Hh signaling-

induced degradation (Chen et al., 2011). Although an increased level of phospho-serine upon

Pthlh treatment was observed in this study, it will be necessary to confirm the phosphorylation

site(s) of Sufu in those Pthlh-treated chondrocytes by examining their Ser-342 and Ser-346

phosphorylation levels. I found that Sufu is required for Pthlh to promote Gli2 degradation and

the processing of Gli3, it is tempting to speculate that phosphorylation of Sufu by PKA stabilizes

Sufu-Gli2/3 complexes, allowing proteolytic processing of Gli2 and Gli3. Further investigation

of this issue requires examination of mice with mutated PKA phosphorylation site(s) of Sufu.

100

Thus, controlling Sufu activity by regulating its protein stability may again serve as a common

mechanism between Ihh and Pthlh signaling in the developing growth plate (Fig. 4-2). In turn

this gradual inactivation of Sufu with chondrocyte differentiation could establish a differential

responsiveness of differentiating chondrocytes to various signaling cues in the growth plate, such

as Ihh itself.

4.2.2 Transcriptional regulation of Sufu

The necessity for proper control of Sufu activity during chondrocyte differentiation raises

the question of whether Sufu activity is also regulated by transcriptional mechanisms in the

growth plate in addition to post-translational regulation. One potential candidate is Sox9, which

is a key regulator for chondrogenesis, chondrocyte proliferation, and expression of cartilage

matrix, such as Col2a1, Col9a1, Col11a1, Aggrecan. Sox9 also inhibits the transition of

chondrocyte into hypertrophic chondrocyte and subsequent endochondral ossification. I

hypothesize that Sox9 inhibits the expression of Sufu at the mRNA level. This hypothesis is

supported by several lines of evidence. In the wild-type growth plate, Sox9 is expressed in

resting and proliferating chondrocytes, with a maximum of expression in prehypertrophic

chondrocyte, but its expression is abolished in hypertrophic chondrocytes (Zhao et al., 1997).

Chondrocyte-specific knockout of Sox9 (Col2a1-Cre; Sox9f/f

) results in severe chondrodysplasia,

which is a characteristic of defective Hh signaling, as well as reduced expression of Ihh and its

downstream target genes, Pthlh and Ptch1 (Akiyama et al., 2002). Furthermore, studies done in

cultured multipotent neural precursor cells show that Sufu is a direct target gene of Sox10, which

together with Sox8 and Sox9 belong to the SoxE family of transcription factors (Pozniak et al.,

101

2010). Examination of Sufu transcripts via in situ hybridization in the Col2a1-Cre; Sox9f/f

mutant

growth plate will provide preliminary information to this hypothesis. Identification of potential

Sox9-binding sites at the promoter region of Sufu through in silico analysis combined with

chromatin immunoprecipitation (ChIP) will be necessary to further determine whether Sox9

modulates Sufu expression level in chondrocytes.

4.2.3 How does Sufu regulate subsequent steps of endochondral ossification?

The lack of Sufu expression in the hypertrophic region of the growth plate is of particular

interest since it suggests that the downregulation of Sufu might be permissive for subsequent

transitional changes from the cartilage template to bone, such as cartilage absorption,

angiogenesis, and trabecular bone formation. At the lower end of the hypertrophic zone in the

growth plate, hypertrophic chondrocytes further mature to become terminally differentiated

chondrocytes, a transition which is marked by upregulation of osteopontin (Farzan et al., 2008),

Vegfa (Gerber et al., 1999), and Mmp13 (Johansson et al., 1997). Vegfa is required for capillary

invasion into the hypertrophic zone in the diaphysis from the perichondrium (Carlevaro et al.,

2000; Gerber et al., 1999; Maes et al., 2004; Zelzer et al., 2002). Mmp13, which is a matrix

metalloproteinase, loosens up the matrix secreted by hypertrophic chondrocytes in order to

facilitate blood vessel invasion (Inada et al., 2004; Johansson et al., 1997; Selvamurugan et al.,

2004). In the Col2a1-Cre; Sufuf/f

mice, a delay in ossification was observed in both the tibial and

vertebral growth plates (Fig. 2-2), suggesting that Sufu may play a role in cartilage

vascularization. It will be interesting to generate transgenic mouse lines misexpressing Sufu in

hypertrophic chondrocytes under the Col10a1 promoter. In situ hybridization and qRT-PCR

102

studies will be needed to demonstrate the level of osteopontin, vegfa and Mmp13 expression in

the growth plate as a result of Sufu overexpression.

4.2.4 Sufu and osteoarthritis

Activated Hh signaling in chondrocytes is associated with predisposition to the

development of osteoarthritis and the level of Hh signaling activity in chondrocytes is associated

with the severity of osteoarthritis (Lin et al., 2009). In this condition, articular chondrocytes

undergo phenotypic and gene expression changes that are reminiscent of those that occur in the

growth plate. Such a process at the articular surface could contribute to the onset of

osteoarthritis.

However, the molecular mechanism underlying the Hh pathway activation in osteoarthritis

development is unclear. I observed the expression of Sufu at the articular surface of wild-type

tibia (Fig. 2-1C). It is tempting to speculate that, similar to what was observed in chondrocyte

hypertrophic differentiation, downregulation of Sufu is required at the articular surface for the

development of osteoarthritis. If this hypothesis is correct, the next question would be does

overexpression of Sufu attenuate the progression of osteoarthritis in mice in vivo? The role of

Sufu and Kif7 in the development of osteoarthritis has not been tested, but can be, as described

below.

Consistent with the notion that Hh signaling activation is associated with a predisposition

to osteoarthritis, reduced Alcian blue staining was observed in the superficial layers of the

articular cartilage of Col2a1-Cre; Sufuf/f

mice and further reduction was observed in the Col2a1-

103

Cre; Sufuf/f

;Kif7f/f

mice at E16.5. Sufu is negatively regulated by Kif7 in chondrocytes. Thus, in

the absence of Kif7, the increased level of Sufu may be involved in maintaining chondrocyte

characteristics at the articular surface. Indeed, Alcian blue-stained cartilage was observed

distributed evenly at the articular surface of Kif7-deficient growth plates at E16.5. The next step

to test the significance of Sufu and Kif7 in developing osteoarthritis would be to look at Sufu

expression level in osteoarthritic cartilage samples from mouse and/or human. The perinatal

lethality in Col2a1-Cre; Sufuf/f

precludes comprehensive studies on the role of Sufu in

osteoarthritis. To investigate whether Sufu inactivation predisposes mice to osteoarthritis,

examination of the joint phenotype of genetically modified mice in which Sufu is deleted

postnatally will be needed. Sufu level is negatively regulated by Kif7 in chondrocytes. Thus, the

effect of overexpression of Sufu on developing osteoarthritis could be tested in Kif7-deficient

mice (Col2a1-Cre;Kif7f/f

). To generate conditional knockout of Sufu, Sufuf/f

mice could be

crossed with Col2a1-rtTA-Cre mice, which express Cre recombinase under Col2a1 regulatory

elements inducible by doxycycline so that Sufu can be deleted in chondrocytes when we

administer doxycycline (Grover and Roughley, 2006; Jeong et al., 2004). Doxycyclin inducible

chondrocyte specific knockout of Sufu and Kif7 (Col2a1-rtTA-Cre;Sufuf/f

;Kif7f/f

) could be

generated in the same manner. Radiographic and histological analysis could be used to assess

the severity of osteoarthritis on wild-type, Col2a1-rtTA-Cre;Sufuf/f

, Col2a1-rtTA-

Cre;Sufuf/f

;Kif7f/f

, and Col2a1-Cre; Kif7f/f

mice at 18-week-old. qRT-PCR analysis would be

needed for genes known to be upregulated in osteoarthritis, such as Adamts5, Col10a1 and

Mmp13 in all samples. Overall, it would be interesting to see if Sufu inactivation in articular

chondrocytes signals the cells to undergo hypertrophic differentiation. If so, can Sufu

104

overexpression help to maintain their chondrocyte characteristics and therefore serve as a

protective mechanism against cartilage degeneration over time?

105

4.3 The role of Sufu in Wnt/ß-catenin signaling pathway during

chondrocyte development

4.3.1 Wnt/ß-catenin signaling in skeletal development

Similar to Ihh and Pthlh signaling, the Wnt/ß-catenin pathway plays an important role in

several aspects of endochondral ossification, such as chondrocyte and osteoblast differentiation.

ß-catenin is a major mediator of Wnt signaling (Kikuchi, 2000; Miller et al., 1999; Waltzer and

Bienz, 1999) which has an important role in chondrocyte differentiation, as demonstrated by the

growth plate phenotype in mutants lacking ß-catenin in their chondrocytes. Studies have shown

that overexpression of ß-catenin inhibits the differentiation of chondrocyte progenitors, enhances

hypertrophic chondrocyte differentiation and promotes the expression of markers for terminally

differentiated chondrocytes, including MMP13 and vegfa (Hartmann and Tabin, 2000; Ryu et al.,

2002). However, the precise function and regulation of ß-catenin in chondrocyte differentiation

still needs to be clarified.

ß-catenin functions as a downstream intracellular signaling molecule in the canonical

Wnt pathway, which regulates cell proliferation and cell differentiation, and cell migration in

many aspects of embryogenesis (Huelsken and Birchmeier, 2001; Moon et al., 2002). A large

multiprotein complex that includes axin and adenomatous polyposis coli gene product (APC)

normally facilitates ß-catenin phosphorylation by glycogen synthase kinase 3ß (GSK3ß) (Ikeda

et al., 1998; Kishida et al., 1999; Liu et al., 2002). Phosphorylated ß-catenin is ubiquitinated and

targeted for degradation through the proteasome-mediated pathway (Kitagawa et al., 1999). In

the absence of Wnt ligand, ß-catenin phosphorylation and subsequent degradation are inhibited.

106

ß-catenin then accumulates in the cell and translocates into the nucleus, where it binds to the

transcription factors T-cell factor (TCF) and lymphoid enhancer factor (Lef), leading to the

activation of pathway target genes. The molecular mechanism underlying the cross-talk between

Ihh, Pthlh, and Wnt/ß-catenin signaling pathways during chondrocyte differentiation remains

elusive. Investigating the genetic interaction between Ihh, Pthlh, and Wnt/ß-catenin signaling

pathways will provide further insight into the means by which different signaling cascades

orchestrate cartilage development.

4.3.2 The role of Sufu in mediating Wnt/ ß-catenin signaling

Sufu and ß-catenin are present in the same complex and Sufu can bind ß-catenin and

export it from the nucleus thereby negatively regulating TCF-dependent transcription in the

human colon cancer SW480 cell line (Meng et al., 2001). Immunofluorescence studies have

shown that whereas much of the endogenous ß-catenin is localized in the cytoplasm of

proliferating and prehypertrophic chondrocytes, nuclear localization of ß-catenin was observed

in hypertrophic chondrocytes (Enomoto-Iwamoto et al., 2002). In chapter 2 I described the

expression pattern of Sufu in the growth plate. Sufu was absent in hypertrophic chondrocytes,

where ß-catenin resides in the nucleus. This result corroborates the notion that Sufu inhibits the

nuclear localization of ß-catenin. Thus, the downregulation of Sufu may be required for ß-

catenin nuclear localization in the hypertrophic chondrocytes. Consistent with an inhibitory role

of Sufu in regulating Wnt/ß-catenin signaling activity, I found a ~40% increase in ß-catenin

levels in Sufu-deficient chondrocytes demonstrated by western analysis. To clarify the role of

Sufu in regulating ß-catenin activity and cellular localization, it will be necessary to examine the

107

expression level of Wnt/ß-catenin pathway target genes by qRT-PCR and to visualize the

subcellular localization of ß-catenin through immunohistochemical or immunofluorescence

analysis in the Sufu-deficient growth plate chondrocytes.

In chapter 3 I showed that treatment of growth plate chondrocytes with Pthlh increases

Sufu protein level through phosphorylation. Interestingly, phosphorylation of ß-catenin at Ser-

675 by PKA promotes ß-catenin stability by inhibiting its ubiquitination (Hino et al., 2005).

Thus, activation of Pthlh/PKA signaling increases the level of ß-catenin, which interacts with

Sufu. I speculate that Sufu plays a role in mediating the stabilization effect of Pthlh on ß-catenin.

To test that, western analysis was performed on lysates derived from wild-type and Sufu-

deficient growth plates with or without Pthlh treatment. As expected, Pthlh treatment resulted in

increased levels of ß-catenin (~2-fold) in chondrocytes. However, this effect of Pthlh on ß-

catenin protein level is absent in the Sufu-deficient cells, suggesting that Sufu is required for the

positive effect of Pthlh on the level of ß-catenin. It is necessary to confirm the role of PKA as a

major effector of Pthlh signaling activation in this process by analyzing the level of ß-catenin in

cells treated with Pthlh and H89, a PKA inhibitor. The mechanism by which Sufu mediates the

stabilization of -catenin is unknown. One possibility is that the Ser-675 residue of ß-catenin

may be masked by changes of its three-dimensional structure in the absence of Sufu.

Alternatively, Sufu may be required for the cytoplasmic localization of ß-catenin where it can be

phosphorylated and stabilized.

Wnt/ß-catenin signaling promotes chondrocyte hypertrophic differentiation, whereas

Pthlh signaling inhibits it. Thus, activation of Pthlh in chondrocytes opposes Wnt/ß-catenin

signaling. Although Wnt/ ß-catenin signaling has been shown to regulate osteoblast

differentiation from the perichondrium at late stage chondrocyte differentiation, the function of

108

high levels of ß-catenin upon Pthlh/PKA stimulation in articular/resting and proliferating

chondrocytes in vivo is unknown. This complicated pattern may allow regulatory cross-talk with

other signaling pathways that are useful. One possible scenario is that Pthlh upregulates ß-

catenin and Sufu levels to ensure sufficient amounts of ß-catenin throughout the course of

chondrocyte hypertrophic differentiation; upregulation of Sufu levels may serve as an important

mechanism by which cells maintain low Wnt signaling such that chondrocytes can differentiate.

Later, in hypertrophic chondrocytes, Sufu inhibition is relieved, ß-catenin translocates into the

nucleus and activates expression of target genes, such as MMP13 and vegfa, which are essential

for subsequent bone formation.

The involvement of Sufu in the regulation of Wnt/ß-catenin signaling provides evidence

that multiple levels of cross-talk exist between Ihh, Pthlh, and Wnt/ß-catenin signaling. It also

highlights the importance of Sufu as a general mediator for multiple signaling pathways during

chondrocyte development. To this end, it will be interesting to further investigate the function of

ß-catenin in Sufu-expressing chondrocytes by generating Col2a1-Cre;Sufuf/f

; Catnbtm1Tak

mice,

which express stabilized ß-catenin in the absence of Sufu in growth plate chondrocytes.

109

4.4 Conclusions

Proper coordinate control of cell proliferation and differentiation through cell-cell

signaling is a major strategy during endochondral ossification. The well-orchestrated cross-talk

between genes spatially and temporally is essential to guide the patterning of each of the skeletal

elements. I demonstrated that the molecular mechanism by which Sufu and Kif7 coordinate Ihh

signaling and further pinpointed the role of Sufu in mediating Pthlh signaling in chondrocytes.

These discoveries suggest that Sufu levels could be critical for cells to respond to multiple

signaling pathways in chondrogenesis. Thus, understanding the mechanism underlying Sufu

regulation may provide insight into cartilage regeneration in vitro. As other signaling pathways,

such as Wnt/ ß-catenin signaling, also play important roles in controlling skeletal development,

further investigation of how Sufu is integrated with various signaling pathways will help us to

gain a full picture of the molecular regulatory network in this process.

110

4.5 Figures

Figure 4-1 Proposed model by which Sufu protein stability serves as a common mechanism that regulates Gli-mediated

transcription.

Figure 4-1. Proposed model by which Sufu protein stability serves as a common mechanism that

regulates Gli-mediated transcription. Sufu protein stability is negatively regulated by Smo-

mediated Hh activation and Kif7 through a proteasome-mediated pathway. Phosphorylation of

Sufu upon Pthlh signaling activation promotes Sufu stabilization resulted in reduced expression

of Hh target genes.

111

Figure 4-2 Proposed mechanism by which Pthlh and Ihh signaling coordinate Sufu expression in the growth plate.

Figure 4-2. Proposed mechanism by which Pthlh and Ihh signaling coordinate Sufu level in the

growth plate. Pthlh positively regulates Sufu expression through phosphorylation, and Ihh

negatively regulates Sufu level thorough promoting its degradation. Sox9 may negatively

regulate Sufu expression at the mRNA level.

112

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116

Appendix A: Pthlh negatively regulates Hh signaling activity

in chondrocytes through a cilia-independent mechanism

Shu-Hsuan C. Hsu, Xiaoyun Zhang, and Benjamin A. Alman

All experiments and analysis described here were conducted by Shu-Hsuan C. Hsu with the

exception of the western analysis by Xiaoyun Zhang.

117

A.1 Summary

Chondrocyte proliferation and differentiation are tightly controlled by Indian hedgehog

(Ihh) and parathyroid hormone-like hormone (Pthlh) signaling pathways in the growth plate.

Pthlh is known to function as a negative regulator of chondrocyte hypertrophic differentiation

and of the Hh signaling pathway. Smo-mediated Hh pathway activation depends on the primary

cilium, which plays a positive role in regulating Hh activity in chondrocytes. Here I show that

Pthlh functions in chondrocytes through a cilia-independent mechanism. The data suggest a

model in which Pthlh promotes Sufu-Gli complexes accumulation at the cilium, resulting in

reduced Hh pathway activity. However, in the absence of cilia, Pthlh treatment resulted in

reduced Hh pathway activity, suggesting that the ciliary tip localization of Sufu-Gli complexes is

not required for their ability to regulate transcription.

118

A.2 Introduction

Cilia are microtubule-based organelles that project from the surface of most eukaryotic

cells (Davenport and Yoder, 2005; Pan et al., 2005; Satir and Christensen, 2007). The cilium

extends from a basal body, which is a centriole-derived microtubule organizing center. Cilia are

assembled and maintained through intraflagellar transport (IFT), a process involving anterograde

and retrograde transportation of IFT particles, mediating by kinesin and dynein motors,

respectively (Bisgrove and Yost, 2006; Davenport and Yoder, 2005; Pan et al., 2005; Satir and

Christensen, 2007). Disruption of the expression and function of IFT particles, such as IFT88,

results in the loss of cilia (Haycraft et al., 2007; Yoder et al., 2002). Ift88ORPK/ ORPK

mouse, which

was developed from a large-scale transgene-induced insertional mutagenesis project at the Oak

Ridge National Laboratory harbors in 1994, exhibits scruffy fur, growth retardation, and

polydactyly (Moyer et al., 1994).

During growth plate development, Indian Hedgehog (Ihh) is secreted by the

prehypertrophic chondrocytes and it regulates chondrocyte hypertrophic differentiation through

upregulating parathyroid hormone-like hormone (Pthlh) expression at the periarticular region

(Chung et al., 1998; St-Jacques et al., 1999; Vortkamp et al., 1996). Pthlh functions to maintain

chondrocytes at the proliferative state and delay hypertrophic differentiation thus reducing the

level of Ihh expression in prehypertrophic chondrocytes (Karaplis et al., 1994; Lanske et al.,

1996). This negative feedback loop formed by Ihh and Pthlh plays an important role in

coordinating chondrocyte proliferation and differentiation in the growth plate (Chung et al.,

1998; Vortkamp et al., 1996).

119

In vertebrates, the effects of Hh signaling are mediated by three Gli transcription factors

(Gli1-3). Upon Hh ligand binding to its receptor, patched 1(Ptch1), Ptch1 inhibition on

smoothened (Smo) is released, leading to activation of Gli-mediated transcription in the nucleus.

Suppressor of fused (Sufu) and kinesin member 7 (Kif7) are two evolutionary conserved

regulators of Gli transcription factors in mammalian Hh signaling (Wilson et al., 2009). During

growth plate development, Sufu acts as a major negative regulator of Gli transcription factors

whereas Kif7 functions both positively and negatively in chondrocytes (Hsu et al., 2011). The

primary cilium serves as the focal point of hedgehog (Hh) signaling regulation in vertebrates.

Important components of the Hh pathway, suppressor of fused (Sufu) and the Gli proteins,

localize to cilia and Smo and Kif7 translocate to cilia upon pathway activation (Corbit et al.,

2005; Haycraft et al., 2005). Recent studies carried out in cultured fibroblasts showed that Smo

plays a positively role in dissociating of inhibitory Sufu-Gli complexes at the primary cilium

upon Hh pathway activation. Therefore a model by which Hh signaling activates Gli protein at

the cilia through relieving the inhibitory function of Sufu, leading to Gli-mediated transcription

activation was proposed.

Studies of explants lacking Gli genes suggest the involvement of Gli3 in the inhibitory

effect of Pthlh on growth plate chondrocyte differentiation. Pthlh represses Gli-mediated

transcription via a protein kinase A (PKA)-dependent mechanism (Mau et al., 2007; Miao et al.,

2004; Vortkamp et al., 1996). These data suggest that besides the Hh-mediated regulation, the

Gli transcription factors may also be regulated by Pthlh signaling. However, the molecular

mechanism as well as the role of cilia in the Pthlh-mediated regulation of Gli activity is

unknown.

120

In this study, I explored the role of primary cilium in regulating the effect of Pthlh on Hh

signaling activity during chondrocytes development using Ift88ORPK/ ORPK

mice. My results

indicate that the primary cilia are not required for the inhibitory effect of Pthlh on chondrocyte

hypertrophic differentiation or Gli-mediated transcription activity. Sufu is localized to the ciliary

tip of resting chondrocytes in vivo upon Pthlh stimulation, which is consistent with previous

studies showing that phosphorylation of Sufu by PKA regulates its ciliary localization (Chen et

al., 2011). However, the ciliary tip localization of Sufu is not required for the negative regulation

of Pthlh on downstream Gli transcription activity.

121

A.3 Results

A.3.1 Ift88ORPK/ ORPK

mice show skeletal defects and dysregulated Hh signaling in

chondrocytes

To investigate the role of primary cilia in growth plate chondrocyte development, I

examined the skeletal phenotype of Ift88ORPK/ ORPK

mice. Consistent with previous studies,

Ift88ORPK/ ORPK

mice survive into young adulthood and exhibit polydactyly and shortening of the

bone in the limbs due to defects in endochondral bone formation (Fig. A-1). Histological analysis

revealed asymmetric chondrocyte hypertrophy as well as a delay in vascularization of the

primary ossification center in Ift88ORPK/ ORPK

mice compared with wild-type littermates (Fig. A-

1). Notably, this asymmetric hypertrophy phenotype does recover with time, and two opposite

growth plates were observed in the tibia of Ift88ORPK/ ORPK

mice at E18.5 (Fig. A-2). In support of

the role of cilia in mediating Hh signaling during development, I found reduced Ptch1 and Gli1

mRNA expression in the cilia-deficient chondrocytes compared with wild-type controls.

Activation of Ptch1 and Gli1 upon Hh ligand stimulation is lost in Ift88ORPK/ ORPK

chondrocytes,

suggesting that cilia are required for Hh ligand responsiveness in chondrocytes (Fig. A-3).

A.3.2 Pthlh downregulates Col10a1 expression and Hh signaling activity through a

cilia-independent mechanism in chondrocytes

In chapter 2 I demonstrated that Pthlh represses Hh signaling activity in part through

increasing Sufu levels and facilitating Gli2 degradation and Gli3R formation in chondrocytes.

122

However, questions remain regarding the role of primary cilia in mediating the effect of Pthlh on

Hh activity regulation. In particular, the involvement of cilia in Pthlh signaling in chondrocytes

needs to be tested. To begin to explore the role of primary cilia in mediating Pthlh signaling

during chondrocyte differentiation, I performed Pthlh treatment on wild-type and Ift88ORPK/ ORPK

micromass cultures at various time points followed by qRT-PCR analysis for Col10a1 and Ptch1,

as readouts for hypertrophy and Hh activity, respectively. The level of Col10a1 expression

increased with time in wild-type cultures, demonstrating chondrocyte hypertrophy in vitro.

Deletion of Ift88 resulted in reduced levels of Col10a1 expression in all three time points

examined (Fig.A-4). Pthlh treatment resulted in a reduction in the Col10a1 expression levels in

differentiated chondrocytes cultures (at Day 6 and 9) from both wild-type and Ift88ORPK/ ORPK

mice (Fig.A-4), suggesting that Pthlh inhibits Col10a1 expression in chondrocytes in a cilia-

independent manner. In wild-type cultures, Ptch1 expression progressively increases with time,

which mimics what was seen during chondrocyte differentiation in the growth plate. In the

absence of Ift88, Ptch1 expression is compromised, although increased Ptch1 expression can still

be observed over time (Fig.A-4). Pthlh treatment resulted in decreased levels of Ptch1 expression

in differentiated chondrocytes in both wild-type and Ift88ORPK/ ORPK

cultures (Fig.A-4). These data

suggest that cilia are not required for the regulation of Gli transcription activity and chondrocyte

hypertrophic differentiation by Pthlh.

A.3.3 Ciliary tip localization of Sufu is induced by Pthlh

In chapter 2 I demonstrated that Kif7 plays a role in the exclusion of Sufu-Gli complexes

from the primary cilium. To investigate whether primary cilium is required for Sufu and Kif7

123

protein and/or RNA expression, I examined Sufu and Kif7 expression in Ift88ORPK/ ORPK

mice. No

significant difference in protein or RNA levels of Sufu and Kif7 was detected in Ift88ORPK/ ORPK

cells (Fig A-5A, B), suggesting that primary cilia are not required for the expression of Sufu and

Kif7. The observations that Pthlh represses Hh signaling activity independent of cilia prompted

us to investigate the ciliary localization of Sufu and Kif7 upon Pthlh signaling activation in the

presence or absence of primary cilium. Studies have shown that Sufu and Gli proteins are

transported into the primary cilium as a complex (Hsu et al., 2011; Humke et al., 2010;

Tukachinsky et al., 2010). In Gli2-/-

; Gli3-/-

embryonic fibroblasts, where all Gli proteins are

absent, Sufu is not detected in the cilia, even upon Shh stimulation, suggesting that Gli proteins

are necessary to recruit Sufu to the cilia (Tukachinsky et al., 2010; Zeng et al., 2010). Thus, the

immunostaining of Sufu could serve as an indicator of Sufu-Gli complexes at the primary cilium.

In resting chondrocytes (where the Hh pathway is inactive), Kif7 was mostly localized to the

ciliary tip, whereas Sufu was found at the basal bodies in ~75% of these cells but was rarely

detected at the ciliary tip (Fig A-5C-E). Both Sufu and Kif7 signals are specific as they are

absent in Sufu-deficient and Kif7-deficient chondrocytes, respectively (Fig. 2-8A). Together,

these results indicate that there is very little Sufu-Gli complexes in the primary cilium of resting

wild-type chondrocytes. Pthlh treatment resulted in a ~20% decrease in the ciliary tip

localization of Kif7 (Fig. A-5C-E). Consistent with recent studies carried out in cultured

fibroblasts showing that PKA phosphorylation of Sufu promotes its ciliary localization (Chen et

al., 2011), Sufu was detected at the ciliary tip in ~50% of the Pthlh-treated wild-type

chondrocytes (Fig. A-5C-E), suggesting that Pthlh stimulates Sufu ciliary tip localization

possibility through promote phosphorylation of Sufu by PKA. Interestingly, in the absence of the

primary cilium, ~80% of resting chondrocytes showed Sufu and Kif7 staining at the basal body

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(Fig. A-5C, F, G). Although no change in the Sufu basal body localization was detected, a ~40%

decrease of Kif7 basal body localization was found upon Pthlh stimulation (Fig. A-5C, F, G).

These observations suggest that Pthlh promotes Sufu-Gli complexes ciliary tip localization in

wild-type chondrocytes, and the ciliary tip localization of Sufu-Gli complexes is not required for

their downstream inhibitory function on Gli-mediated transcription activity. In chapter 3 I

demonstrated that Kif7 is not required for the negative effect of Pthlh on growth plate

chondrocytes. Thus, the changes in Kif7 ciliary localization may play a minor role in regulating

the effect of Pthlh on chondrocyte Hh signaling activity.

125

A.4 Discussion

In chapter 2 I demonstrated distinct and overlapping functions of Sufu and Kif7 in Hh

signaling during chondrocyte development. In chapter 3 I established that Sufu plays an

important role in regulating the negative effect of Pthlh on chondrocyte hypertrophic

differentiation, Gli proteins processing, and Hh signaling activity. There is evidence suggesting

that the primary cilium serves as a focal point in Hh signaling regulation in vertebrates. Here, I

provided data to indicate that Pthlh functions to inhibit chondrocyte hypertrophic differentiation

and Hh signaling activity in chondrocytes through a cilia-independent mechanism.

Phenotypic analysis of Ift88ORPK/ ORPK

mice indicates that the primary cilia are required

for normal skeletal development. Ift88ORPK/ ORPK

chondrocytes showed reduced Hh pathway

activity and lack of response to Hh ligand stimulation compared to wild-type controls. Pthlh

possess inhibitory effect on the expression of Col10a1 and Ptch1 in wild-type micromass

cultures. This is consistent with its negative role in regulating chondrocyte hypertrophic

differentiation and Hh signaling activity in the growth plate in vivo. Similarly, reduced Col10a1

and Ptch1 expression were observed upon Pthlh signaling activation in cilia-deficient (Ift88ORPK/

ORPK) micromass cultures. The data suggest that primary cilia are not required for Pthlh ligand

responsiveness or the negative effect of Pthlh on chondrocyte differentiation and Hh activity

regulation.

Hh pathway activation stimulates degradation of Sufu in some cancer cell lines (Yue et

al., 2009). Recent studies suggest that Hh stimulation promotes the dissociation of Sufu-Gli

protein complexes at the ciliary tip and that this dissociation is important for Gli activation

(Humke et al., 2010; Tukachinsky et al., 2010). In chapter 3 I showed that Pthlh plays a positive

126

role in controlling the stability of Sufu protein as well as regulating Gli2 protein degradation and

Gli3 repressor (Gli3R) formation in the growth plate chondrocytes. Interestingly, here I found

that Sufu is mostly excluded from the ciliary tip in wild-type chondrocytes, whereas Sufu

appears to be stabilized in the ciliary tip in the presence of Pthlh. This is consistent with the

observation that Hh pathway activity is reduced in Pthlh-treated cells, owing to inefficient

dissociation of the Sufu-Gli complexes. Sufu is found to be localized at the basal body in the

absence of the primary cilium and there is no change in its basal body localization observed upon

Pthlh activation. The data suggest that the ciliary tip localization is not required for the inhibitory

effect of Sufu-Gli complexes in chondrocytes. However, whether the ciliary tip localization

contributes to the stabilization of Sufu in Pthlh-treated chondrocytes or its related to the

increased level of Sufu protein awaits further investigation. Nevertheless, results here

demonstrated that Pthlh functions independent of primary cilia in chondrocytes.

Previous in vitro studies show that Hh ligand stimulation promotes Kif7 ciliary tip

translocation (Liem et al., 2009). In chapter 2 I found that Kif7 localizes to the ciliary tip in the

proliferating chondrocytes in vivo. Here I reported that Kif7 localizes also to the ciliary tip in the

resting chondrocytes where the Hh pathway is inactive (as indicated by the lack of Hh target

gene expression). These results suggest that Kif7 might have a functional role in the primary

cilium, even in the absence of Hh ligand activation which supports the proposed model in

chapter 2 (Fig. 2-10B). In the cilia-deficient (Ift88ORPK/ ORPK

) chondrocytes, Sufu localizes to the

basal body in the absence or presence of Pthlh, suggesting that the ciliary tip localization is not

the key control for the inhibitory function of Sufu-Gli complexes, and their ability to regulate

transcription. This notion is consistent with the previous reports showing that Sufu functions

independent of the primary cilium in controlling Gli protein levels (Chen et al., 2009). Taken

127

together, the data suggest that despite the importance of the primary cilium in controlling Hh

pathway activation, the primary cilium is not required for Sufu-mediated negative regulation of

Hh pathway activity in chondrocytes.

Recent studies have shown that PKA, the main mediator of Pthlh signaling in

chondrocytes, localizes to the basal body in cultured fibroblasts (Barzi et al., 2010; Tuson et al.,

2011). This colocalization of PKA and Sufu prompts me to propose a model for cilia-mediated

regulation of Gli activity (Fig. A-6). In the presence of Hh ligand, Sufu-Gli complexes enter the

cilium and are modified within the cilium, either by covalent modification or through interaction

with other proteins such as Kif7, to allow dissociation of the complexes and activation of the Hh

pathway. In the presence of Pthlh ligand, Sufu is phosphorylated by PKA; p-Sufu-Gli complexes

enter the cilium. After exiting the cilium Gli2 complexed with Sufu is recognized and

phosphorylated by PKA, which leads to Gli2 degradation. Phosphorylation by PKA targets Gli3

complexed with Sufu to proteasome-mediated processing into Gli3R.

Taken together, I show here a cilia-independent mechanism by which Pthlh mediates

chondrocyte hypertrophic differentiation and Hh signaling activity regulation in chondrocytes.

Therefore, I propose that in wild-type chondrocytes, Pthlh inhibits hypertrophic differentiation

and Gli-mediated transcription. There is an increased level of Sufu-Gli complexes in the primary

cilium upon Pthlh activation, resulting in an inhibition of Hh signaling. In the absence of the

primary cilium, the inhibitory function of Sufu-Gli complexes remains. Pthlh treatment results in

an increased level of Sufu-Gli complexes localized at the basal body, resulting in reduced Hh

pathway activity.

128

A.5 Materials & Methods

Ethics Statement



Mice

Ift88ORPK

(Tg737ORPK

) mutants were received from Dr. Yoder (The University of

Alabama). The mice were genotyped as described previously (Yoder et al., 1996). In all cases,

littermate mice were used as controls. A mouse protocol describing the above experimental

procedures was approved by the Animal Care Committee of The Hospital for Sick Children.

Skeletal staining







129






minimize morphological variations due to anatomic location. Hematoxylin and eosin (H&E)

staining were performed using standard techniques.

Real-time quantitative PCR




mouse Gli1, Ptch1, and Col10a1 (Applied Biosystems). The gene expression levels between

samples were analyzed using the 2 ΔΔCt

method (Livak and Schmittgen, 2001). Either GAPDH

or β-actin (Applied Biosystems) was used as endogenous control for target gene normalization.


Chondrocyte isolation protocol was modified from previous published methods (Gosset




130

clean petri dish with 0.5 mg/ml collagenase type 4 solution and were incubated overnight at

37°C. Collagenase type 4 solution containing chondrocytes was collected. Cells were washed

with PBS and seeded at a density of 8x103 cells per cm

2.

Micromass cultures

Fore- and hind-limbs were isolated from E11.5 mouse embryos and collected in growth

medium (DMEM + 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin),

washed twice in PBS, and digested in 0.1% trypsin and 0.1% collagenase D (Worthington

Biochemical) in PBS at 37 °C for 20 min. 20 μl of a single cell suspension (2 × 107 cells/ml) was

plated in the center of 9.6 cm2 dishes. The cultures were incubated at 37 °C in a humidified

incubator with 5% CO2 for 1 h, after which they were gently flooded with 2 ml of growth

medium.

Immunofluorescence

Samples were fixed in 4% paraformaldehyde overnight, embedded in paraffin and

sectioned. Sections (5 µm) of growth plate were cut parallel to the longitudinal axis of the bone.

The sections were stained with a monoclonal antibody against acetylated α-tubulin (clone 6-11b-

1, 1:1000; Sigma-Aldrich, Steinheim, Germany), and/or a monoclonal antibody against γ-tubulin

(Dyomics647) (1:100; Abcam), and/or a monoclonal antibody against Sufu (1:100; Abcam),

and/or a polyclonal antibody against Kif7 (1:100) at 4°C overnight. Secondary antibodies

conjugated with Alexa Fluor 488 (1:100; Invitrogen) and TRITC (1:100 Jackson

131

ImmunoResearch Laboratories) were applied for 45 minutes to detect the primary antibody.

Finally the sections were mounted in 4 ,6-diamidino-2-phenylindole (DAPI) containing

Vectashield (Vector Laboratories). Confocal images were acquired using a spinning disc

confocal microscope.



calculated for each condition, and Student’s t test was utilized to compare data sets. A p value of

< 0.05 was used as a threshold for statistical significance.

132

A.6 Figures

Figure A- 1 The primary cilia are required for normal embryonic skeletal development.

Figure A-1. The primary cilia are required for normal embryonic skeletal development. Upper

panel: Alcian blue/Alizarin red stained hind limbs from E16.5 wild-type and Ift88ORPK/ ORPK K

mice. Note polydactyly (arrowhead) and shortening of the long bones in the Ift88ORPK/ ORPK

limb.

Lower panel: H&E staining showed asymmetrical chondrocyte hypertrophy and delayed in

primary ossification center formation in the Ift88ORPK/ ORPK

mice compared to wild-type

littermates at E16.5.

133

Figure A- 2 Asymmetrical hypertrophy phenotype in Ift88ORPK/ ORPK mice is not seen at E18.5.

Figure A-2. Asymmetrical hypertrophy phenotype in Ift88ORPK/ ORPK K

mice is not seen at E18.5.

Histological and immunohistochemical analysis for Col10a1 expression of the Ift88ORPK/ ORPK K

mouse tibia showed the asymmetrical hypertrophy phenotype observed at E16.5 is not seen at

E18.5.

134

Figure A- 3 The role of primary cilia in chondrocyte Hh ligand responsiveness.

Figure A-3. The role of primary cilia in chondrocyte ligand responsiveness. Reduced level of

Ptch1 and Gli1 expression was found in cilia-deficient chondrocytes assessed by qRT-PCR. Shh

treatment resulted in increased Ptch1 and Gli1 mRNA expression in wild-type chondrocytes. In

cilia-deficient chondrocytes, Shh treatment did not result in Hh signaling activation. Data are

shown as means with 95% confidence intervals indicated (n=3). *P<0.05.

135

Figure A- 4 The role of primary cilia in mediating Pthlh signaling in chondrocyte differentiation.

Figure A-4. The role of primary cilium in mediating Pthlh signaling in chondrocyte

differentiation. Expression of Col10a1 and Ptch1 in micromass cultures of wild-type and

Ift88ORPK/ ORPK K

mice at various time points (Day3, Day6, and Day9) with or without Pthlh

treatment was assessed by qRT-PCR. Data are shown as means with 95% confidence intervals

indicated (n=2). *P<0.05.

136

Figure A- 5 Increased Sufu ciliary localization in Pthlh-treated wild-type chondrocytes.

137

Figure A-5. Increased Sufu ciliary localization in Pthlh-treated wild-type chondrocytes. (A) Sufu

and Kif7 protein levels in wild-type and Ift88ORPK/ ORPK

mice were assessed by western analysis.

Actin was used as loading control. (B) Sufu and Kif7 mRNA expression levels in Ift88ORPK/ ORPK

mice were found to be comparable with wild-type controls. Data are shown as means with 95%

confidence intervals indicated (n=3). (C) Fluorescence micrographs of cilia or basal bodies from

wild-type and Ift88ORPK/ ORPK

growth plate chondrocytes in the present or absence of Pthlh

treatment. Sufu (ciliary tip localization of Sufu is indicated by white arrow; basal body

localization of Sufu is indicated by white asterisk) and Kif7 (ciliary tip localization of Kif7 is

indicated by green arrow; basal body localization of Kif7 is indicated by green asterisk) are

detected in the green channel. ilia are detected by staining against acetylated α-tubulin (red

channel). Centrioles are detected by staining against γ-tubulin (blue channel) to label the base of

the cilia. (D-G) 25 to 50 ciliated cells from the resting zone of wild-type and Ift88ORPK/ ORPK

growth plates with (+Pthlh) or without (Un: untreated) Pthlh treatment were examined. Data are

shown as percentages.

138

Figure A- 6 A proposed model for cilia-mediated regulation of Gli activity.

Figure A-6. A proposed model for cilia-mediated regulation of Gli activity. In this model, PKA-

mediated Gli processing is determined by the appropriate Gli substrate, which becomes available

only when Gli is complexed with phosphorylated Sufu (p-Sufu) in the cytoplasm. In the presence

of Hh ligand, active Smo localizes to the cilia and dissociation of Sufu-Gli complexes in the

cilium is promoted by Smo and Kif7; free Gli proteins enter the nucleus and activate the

transcription. In the presence of Pthlh ligand, Sufu is phosphorylated by PKA and p-Sufu-Gli

complexes enter the cilium. Gli2 complexed with p-Sufu is recognized and phosphorylated by

PKA leading to Gli2 degradation. Gli3 complexed with p-Sufu is targeted for phosphorylation

and proteasome-mediated processing into Gli3R.

139

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